BRPI0807739A2 - COMPOSITION FOR TREATMENT OF CARTILAGE DISEASES - Google Patents

Description

COMPOSITION FOR TREATMENT OF CARTILAGE DISEASES

TECHNICAL FIELD

The present invention relates to a composition for cartilage regeneration or treatment of cartilage disease, including veterinary applications.

BACKGROUND OF THE INVENTION For example, articular cartilage is a hyaline cartridge that is composed of a small number of cells, collagenous extracellular matrix, abundant proteoglycans and

Water. In the case of bones, as there are vascular and neural networks and the bone has the ability to self-regenerate, even if a fracture has occurred, the fracture is often repaired completely. However, the articular cartilage has no vascular and neural networks.

Consequently, it has virtually no potential for self-regeneration and, in the case of formation of large cartilage defects in particular, the cartilage defect is not properly repaired. Even in those portions that are repaired, the fibrous cartilage that is

2 0 formed has different mechanical properties in relation to

to hyaline cartilage. Consequently, when a cartilage defect is formed, joint pain and loss of function often progress to osteoarthritis. In addition, a cartilage defect can vary widely.

as a result of symptoms progressing from the early stages of osteoarthritis that begin with wear of the articular cartilage surface as a result of aging or overuse of the joint.

Thus, as the articular cartilage does not have

With adequate self-healing ability, surgical procedures are required to treat cartilage injuries, examples of which include mosaicplasty, microfracture, perforation, abrasion, and debridement. Among these, microfracture, perforation, and abrasion are called 5 marrow stimulation techniques, and promote bone marrow bleeding to induce bone marrow-derived cartilage precursor cells in anticipation of their differentiation into cartilage. However, these techniques have limitations with respect to cartilage defects 10 that cover large areas, and cartilage regenerated by these methods is in the form of fibrous cartilage that has different mechanical properties than hyaline cartilage.

Peterson et al and Grande et al tested an autologous chondrocyte (ACI) implantation technique on rabbit partial thickness articular cartilage in 1984. ICA is a technique that involves collecting and culturing tissue from a patient's own normal cartilage, implanting cultured cells in an affected area while suspended in a medium, and covering the cartilage defect with the periosteum to prevent extravasation of cells. The ICA was first applied in 1994 and is currently in practice for over 15 years. Several successful results have been reported. However, recent clinical studies report that ICA does not yield significantly superior results when compared to other techniques for repairing articular cartilage defects.

There are two main reasons for these unfavorable results obtained with the ICA. 0 first is the difficulty

The technique associated with the attachment of cells and structure to the cartilage defect and the covering of the defect with a periosteum flap. The ICA technique requires extensive exposure of the joint arthrotomy to periosteum flap suture to cover the cell suspension. In addition, several complications associated with periosteum flap have been reported, including periosteum hypertrophy, defect formation, and intra-articular adhesion. The other reason involves limitations in the use of chondrocytes. Chondrocytes rapidly lose their differential phenotype in cultures of

monolayers, turning into fibroblasts. Another problem is that while ICA requires cartilage to be collected from an unweighted joint site, donor sites remain problematic as chondrocytes are collected from these sites.

On the other hand, attempts are underway to

use natural polymers such as collagen, chitosan, agarose and alginic acid in the regenerative therapy of articular cartilage. In particular, alginic acid is a polysaccharide extracted from algae

20 browns such as Ecklonia, Eisenia and Laminaria,

which has the cross-linking property after the addition of calcium or other divalent metal ions, and attempts have been made to apply alginic acid to injured sites using this property to encrust

cells such as chondrocytes, growth factors, and so on in such a gel (see, for example, References 1, 2, 3, 4, and 5).

For example, Reference 1 discloses an alginate gel comprising a mixture of a soluble alginate and a

Insoluble alginate / gel, while References 2, 3 and 4 disclose the use of alginate globules. In Reference 2, alginic acid may be used as a vehicle that does not impart any disadvantageous effects to an injured site, although alginic acid itself is discussed as having no therapeutic effect. In addition, Reference 4 reveals that the fusion of alginate globule-embedded chondrocytes in host tissue after transplantation into a rabbit cartilage defect was not observed. In addition, although alginate blood cells need to be pressure-applied to a defect, as it is necessary to make the globules match the size of the defect, their use in clinical practice is technically difficult. Reference 5 discloses a graft in which chondrocytes are suspended in sodium alginate and injected into a rabbit cartilage defect, followed by surface curing with a CaCl2 solution in which, although normal cartilage tissue is formed, cartilage forms. fibrous if only alginic acid is applied to the cartilage defect without the presence of cells.

In addition, research is underway on the use of collagen sponge and the like as a cell harness as an example of attempts to use mesenchymal stem cells for regenerative cartilage therapy. Although methods involving mesenchymal stem cell transplantation after in vitro differentiation into chondrocytes and methods involving undifferentiated mesenchymal stem cell transplantation have been considered, there is still an ongoing debate about which method of use is optimal (Reference 6). To the extent that cartilage defects in osteoarthritis (OA) occur widely and in load-bearing regions, their repair by transplantation or regenerative therapy is considered to be difficult. Those 5 cartilage defects eligible for cell transplant cartilage regeneration as described above are limited to partial cartilage defects caused primarily by sports activity or trauma. Treatment of osteoarthritis focuses primarily on the removal of pain and inflammation in the affected area, and it is commonly treated abroad with the administration of non-steroidal anti-inflammatory drugs. However, since renal function may be impaired in elderly patients, continuous oral administration of non-steroidal anti-inflammatory drugs 15 may be difficult from a safety point of view. Products that incorporate hyaluronic acid, which is a component of cartilage synovial fluid, increase the lubricating function of joints when administered to a joint and, as

2 0 that these products also have analgesic action, they are

widely used as agents for improving joint function for osteoarthritis. However, since ultimately there is no choice but to replace the joint with an artificial joint in severe cases of osteoarthritis in which joint damage has progressed, there is a desire for the development of a new therapeutic drug.

REFERENCES

1. International Publication WO 2006/044342.

2. Cay M. Mierisch et al., "Transforming Growth

3 0 Factor β in Calcium Alginate Beads for the Treatment of Articular Cartilage Defects in the Rabbit ", The Journal of Arthroscopic and Related Surgery, Vol. 18, No. 8 (October), 2002: pages 892-900.

3. David R. Diduch et al, "Marrow Stromal Cells Embedded in Alginate for Repair of Osteoehondral Defeets",

The Journal of Arthroscopic and Related Surgery, Vol. 16, No. 6 (September), 2000: pages 571-577.

4. David R. Didueh et al, "Chondrocyte Transplantation into Articular Cartilage Defeets with Use

of Calcium Alginate: The Fate of the Cells, "J. Bone Joint Surg. Am. 85: 2,003, pages 1,757-1,767.

5. E. Fragonas et al, "Articular Cartilage Repair in Rabbits by Using Suspensions of Allogenic Chondrocytes in Alginate", Biomaterials, Vol. 21, 2000: pages 795-801.

6. "Life Science Report No. 4", 2005 (Editor:

"Intellectual Property Department, Tokyo Medical and Dental University", Editor: Maruzen Co., Ltd.), pages 235-243, Contributing Editor: Ichiro Sekiya.

DISCLOSURE OF INVENTION

2 0 Problems to be solved by the invention

When considering the actual clinical applications for regenerative cartilage defect therapy in this way, there was no regenerative therapy capable of being compatible with practical use in terms of problems such as,

for example cytotoxicity, bioaffinity, ease of application and therapeutic effects. Specifically, there was a need for the development of a composition for cartilage regeneration and treatment of cartilage diseases, and a method of treatment with its use.

30 which has superior practicability by overcoming problems in the field of cartilage regenerative therapy by being able to effectively promote cartilage regeneration without requiring an excessive surgical procedure such as the ICA having a simple procedure, and 5 do not place a excessive burden on the body in terms of chondrocyte collection, periosteum and the like, is capable of being used over a wide range of cartilage injury forms, regardless of application conditions, reducing the detrimental effects of

crisscrossing and the like applied to cartilage lesions and having superior bioaffinity. In particular, it should still be a composition capable of regenerating hyaline cartilage using only polymer, without embedded cells.

Although osteoarthritis is a degenerative disease in the

which articular cartilage is worn out due to aging and overuse of the joint, as well as the mechanical cause of the wear, local inflammatory responses, for example, the production of inflammatory cytokines by

2 0 synovial cells and chondrocytes and substance induction

algesics and proteases by inflammatory cytokines are also considered to be involved in joint destruction. Specifically, associated with joint cartilage wear (mechanical damage), a response is induced

Within the joint tissue, self-destructive cartilage damage progresses as a result of this inflammatory response, and mechanical damage progresses further as a result of decreased joint function, thus resulting in a vicious circle that exacerbates

3 0 further the disease. Thus, therapeutic drugs for osteoarthritis are needed that provide comprehensive effects, including effects that protect cartilage against wear, effects that inhibit degenerative changes in cartilage caused by wear and inflammation, effects that repair cartilage trauma injury, and effects that suppress inflammation and pain. If a drug capable of inhibiting inflammation and suppressing joint pain were obtained, it would be applied to treat frozen shoulder and suppress joint pain in chronic rheumatoid arthritis. Hyaluronic acid is inherently an important component of synovial fluid, and its replacement improves joint function. There are currently no drugs that have been shown to have comprehensive therapeutic effects on cartilaginous tissue other than hyaluronic acid. Hyaluronic acid preparations are produced by animal tissue extraction or fermentation, and we seek new materials that can be produced more easily and offer a higher level of safety. In addition, although hyaluronic acid preparations require five consecutive weekly administrations initially, followed by repeated repeated administrations, a new composition that has a longer duration and greater therapeutic effects is sought in order to reduce the number of injections into the joint. knee.

Means for troubleshooting

The inventors of the present invention have performed intensive studies to solve the problems mentioned above. As a result, it has been found that upon application to a cartilage trauma injury of a composition containing a monovalent alginic acid metal salt for which its endotoxin level has been reduced to a degree that does not substantially induce fever or inflammation. , which has a viscosity of 400 to 20,000 mPa.and has fluidity, cartilage regeneration can be promoted by a simple technique without the need for an excessive surgical procedure.

When this composition was applied to a joint cartilage defect and a CaCl2 solution was applied to its surface, the composition did not leave the applied site. Thus, it has been surprisingly found that the composition can be applied even to places subjected to loading under extremely harsh movement conditions such as articular cartilage. Because the viscosity of the composition of the present invention is about 2,000 mPa.s or more, the composition can be applied even if the injured surface is facing downwards.

The composition of the present invention has resulted in extremely superior cartilage regeneration in the

2 0 case of mesenchymal stem cell encrustation of the

bone marrow or stromal cells in this composition. Furthermore, even if such cells are not fouled, it has been found that the composition of the present invention allows satisfactory hyaline cartilage regeneration by hyaline chondrocytes to be achieved, thereby leading to the completion of the present invention.

In addition, it was found that degenerative cartilage changes were inhibited and that the protective effects of cartilage were obtained by application to a

Cartilage trauma injury in an osteoarthritis model of a composition containing a monovalent metal salt of alginic acid for which its endotoxin level has been reduced to a degree that does not substantially induce fever or inflammation. In addition, it has also been found that this composition has pain suppressing effects in an experimental model of arthritis pain, thus leading to the completion of the present invention.

This is the first time that a substance, other than hyaluronic acid, which is an important component of synovial fluid, has been shown to have compound effects on cartilaginous tissue in this way. It was surprising to find that alginic acid, which is a polymer that originates from algae and is not inherently present in animals, has effects like these.

Specifically, the present invention provides the

following composition for cartilage regeneration that is applied to a cartilage trauma injury.

(1-1) A composition that is used for cartilage regeneration and is cured in an area affected by application.

20 in a cartilage trauma injury, which contains a lower endotoxin monovalent metal salt of alginic acid, which has a viscosity of 400 mPa.s to 20,000 mPa.s, and which has fluidity.

(1-2) The composition described in (1-1) above, wherein the monovalent metal salt of alginic acid is sodium alginate.

(1-3) The composition described in (1-2) above, wherein the sodium alginate is sodium alginate having an average weight average molecular weight of 500,000 or more as determined by gel filtration chromatography. (1-4) The composition described in any one of (1-1) to (1-3) above, wherein the application to cartilage trauma injury is: a) application to a cartilage defect, or b) application to the holes after the formation of one or more of the holes in a cartilage trauma injury or cartilage defect.

(1-5) The composition described in any one of (1-1) to (1-4) above, which does not contain cells for regeneration of cartilaginous tissue.

(1-6) The composition described in any one of (1-1) to

(1-4) above, which include cells for regeneration of cartilaginous tissue.

(1-7) The composition described in (1-6) above, wherein, prior to application to a cartilage trauma injury, the composition containing a monovalent alginic acid metal salt having cells embedded therein is soaked. with cells grown in vitro in one or more states selected from the group consisting of: a) a state in which

the cell number is 1 x 106 cells / mL or more, b) a state in which hyaline-like cartilaginous tissue is detected by safranin-0 staining or H-E staining, c) a state in which it is detected type II collagen by anti-collagen II antibody or genetic analysis, d) a state in which agglican is detected by anti-agglutic antibody or genetic analysis, and e) a state in which the extracellular matrix (collagen, hyaluronic acid, proteoglycan) is secreted .

(1-8) The composition described in (1-6) or (1-7) above, wherein cells for regenerating cartilage tissue 30 include bone marrow mesenchymal stem cells. (1-9) The composition described in any one of (1-1) to (1-8) above, wherein the composition is adhered to an injured site for at least 5 seconds when applied to a traumatic injury. cartilage in the state in which an opening in the cartilage defect or an opening in the holes formed in a cartilage trauma injury or cartilage defect is tilted or turned downward.

(1-10) The composition described in any one of (1-1) to (1-9) above, wherein application to a cartilage trauma injury is possible with a 16G needle.

(1-11) The composition described in any one of (1-1) to (1-10) above, wherein the composition is applied to a cartilage trauma injury, and a criss-crossing agent is applied to the surface of the composition. .

(1-12) The composition described in (1-11) above, wherein the

Crisscrossing agent is a CaCl2 solution.

(1-13) The composition described in any one of (1-1) to (1-12) above, wherein the cartilage trauma injury is injured articular cartilage.

(1-14) The composition described in any one of (1-1) to

(1-13) above, wherein cartilage regeneration is for the purpose of regenerating hyaline cartilage.

In addition, the present invention provides a composition which enables therapeutic effects to be obtained by injection into a joint of a patient having a cartilage disease.

(2-1) A composition which is used for the treatment of cartilage disease and which is injected into a joint which contains as an active ingredient therein a monovalent metal salt of lower endotoxin alginic acid.

(2-2) A composition which is used for the inhibition of degenerative cartilage changes and which is injected into a joint containing as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(2-3) A composition which is used for cartilage protection and which is injected into a joint, which contains, as an active ingredient in it, a monovalent metal salt of alginic acid lower endotoxin.

(2-4) A composition which is used for cartilage repair and which is injected into a joint, which contains, as an active ingredient in it, a monovalent metal salt of alginic acid lower endotoxin.

(2-5) A composition that is used for pain suppression

which is injected into a joint which contains, as an active ingredient therein, a monovalent metal salt of lower endotoxin of alginic acid.

(2-6) A composition which is used for inhibiting joint inflammation and which is injected into a joint which contains as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(2-7) A composition which is used for joint function improvement and which is injected into a joint which contains as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(2-8) A composition which is used for the treatment of osteoarthritis and which is injected into a joint, which contains as an active ingredient thereof a monovalent lower salt endotoxin metal salt of alginic acid. (2-9) A composition which is used for the treatment of frozen shoulder and which is injected into a joint, which contains, as an active ingredient in it, a lower endotoxin monovalent metal salt of alginic acid.

(2-10) A composition that is used for suppression of

joint pain associated with rheumatoid arthritis and which is injected into a joint, which contains, as an active ingredient in it, a monovalent metal salt of lower endotoxin from alginic acid.

(2-11) A composition for intra-articular injection that

has the effect of alleviating, ameliorating or curing symptoms associated with cartilage disease, which contains as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(2-12) The composition described in (2-11) above, wherein the

The relief, amelioration or cure effect of symptoms associated with cartilage disease is at least one effect selected from the group consisting of inhibition of degenerative cartilage changes, cartilage protection, cartilage repair, joint pain suppression, inhibition of joint inflammation, and improvement of joint function.

(2-13) The composition described in any one of (2-1) to (2-12) above, wherein the monovalent metal salt of alginic acid is sodium alginate.

(2-14) The composition described in (2-13) above, wherein the

Sodium alginate is sodium alginate having an average molecular weight of 500,000 or more as determined by gel filtration chromatography.

(2-15) A composition that is used for the treatment of

The cartilage disease which is injected into a joint containing as an active ingredient thereof a lower endotoxin sodium alginate having a weight average molecular weight of 500,000 or more as determined by gel filtration chromatography.

In addition, the present invention also provides the

following method of treatment for a cartilage trauma injury and a composition used in that method of treatment.

(3-1) A method of treating a trauma injury

A cartilage composition comprising: applying to a cartilage trauma injury a composition containing a lower endotoxin monovalent metal salt of alginic acid having a viscosity of 400 mPa.s to 20,000 mPa.s and having fluidity.

(3-2) The method described in (3-1) above, wherein the

A composition containing a monovalent metal salt of alginic acid is applied to a cartilage trauma injury, and a crosslinking agent is applied to the surface of the composition to cure the composition.

20 (3-3) The method described in (3-1) or (3-2) above, in

cells for regeneration of cartilaginous tissue are embedded in the composition containing a monovalent metal salt of alginic acid, and the composition is applied to a cartilage trauma injury.

(3-4) The method described in any of (3-1) to (3-

3) above, wherein cells for cartilaginous tissue regeneration are embedded in the composition containing a monovalent alginic acid metal salt, and the composition is applied to a cartilage trauma injury after cultivation.

30 in vitro in one or more states selected from the group consisting of: a) a state in which the number of cells is

1 x 106 cells / mL or more, b) a state in which hyaline-like cartilaginous tissue is detected by safranin-0 staining or HE staining, c) a state in which type II collagen is detected by anticollagen II antibody or genetic analysis, d) a state in which agglican is detected by anti-agglutic antibody or genetic analysis, and e) a state in which the extracellular matrix (collagen, hyaluronic acid, proteoglycan) is

secret.

(3-5) The method described in any of (3-2) to (3-

4) above, wherein the crosslinking agent is a CaCl2 solution.

(3-6) The method described in any of (3-1) to (3-

5) above, wherein the application to a cartilage trauma injury is: a) application to a cartilage defect, or b) formation of one or more holes in the cartilage trauma injury or a cartilage defect and application to the holes formed.

20 (3-7) A composition used in the method described in

any one of (3-1) to (3-6) above, which contains an alginic acid lower endotoxin monovalent metal salt, which has a viscosity of 400 mPa.s to 20,000 mPa.s and is flowable.

(3-8) The composition described in (3-7) above, wherein the

A composition containing a monovalent metal salt of alginic acid contains cells for regeneration of cartilaginous tissue.

(3-9) The composition described in (3-7) or (3-8) above,

30 wherein the monovalent metal salt of alginic acid is sodium alginate.

(3-10) A composition for the treatment of a cartilage injury which contains a monovalent metal salt of alginic acid for which its endotoxin level has been reduced to a degree that does not substantially induce inflammation or fever, which It has a viscosity of 400 mPa.se to 20,000 mPa.se and has fluidity, wherein the composition is used arthroscopically for its application to sufficiently fill the total void of an affected area that is a site of application of a lesion. After cartilage trauma that was previously irrigated and dried, a CaCl2 solution is applied to the surface of the applied composition, followed by removal of the CaCl2 solution that remains on the surface of the applied composition, and the composition is cured in the affected area.

Furthermore, the present invention provides a method of treatment for a cartilage disease and its associated symptoms.

(4-1) A method of treating a disease of the

20 The cartilage comprising: injection into a joint of

a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-2) A method of inhibiting degenerative cartilage changes which comprises: injecting into a joint a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-3) A method of cartilage protection that

30 comprises: injecting into a joint a composition containing as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(4-4) A method of cartilage repair comprising: injecting into a joint a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-5) A method of suppressing joint pain comprising: injecting into a joint a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-6) A method of inhibiting joint inflammation comprising: injecting into a joint a composition containing an alginic acid lower endotoxin monovalent metal salt as an active ingredient thereof.

(4-7) A method of improving joint function comprising: injecting into a joint a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-8) A method of treating osteoarthritis that

comprises: injection into a joint of a composition containing as an active ingredient thereof a monovalent metal salt of lower endotoxin alginic acid.

(4-9) A method of treating a frozen shoulder comprising: injecting into a joint a composition containing an alginic acid lower endotoxin monovalent metal salt as an active ingredient thereof.

(4-10) A method of suppressing joint pain associated with rheumatoid arthritis comprising: injection into

30 is a joint of a composition containing as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

(4-11) The method described in any of (4-1) to (4-10) above, wherein the monovalent metal salt of alginic acid is sodium alginate.

(4-12) The method described in (4-11) above, wherein the sodium alginate is sodium alginate having an average weight average molecular weight of 500,000 or more as determined by gel filtration chromatography.

(4-13) A method for treating a disease of the

cartilage comprising: injecting into a joint a composition containing as an active ingredient thereof a lower endotoxin sodium alginate having a weight average molecular weight of 500,000 or more as

determined by gel filtration chromatography.

Effects of the invention

As the cartilage regeneration composition of the present invention can be injected into a cartilage trauma injury without the need for a surgical procedure.

excessive, the procedure is simple. Cartilage regeneration, and particularly hyaline cartilage regeneration, can be effectively promoted without placing an excessive burden on the body in terms of chondrocyte collection, periosteum, and the like.

2 5 The composition for cartilage regeneration of

The present invention has gel curability as a result of contacting Ca ions in the affected area. The composition may be retained in the affected area by curing its surface by benefiting from this property.

In the case of cells embedded for cartilage tissue regeneration in the composition of the present invention, the cells are easily dispersed in the cured gel. Thus, the composition can be used for various forms of cartilage injury, and is capable of accommodating various application conditions.

The composition for treating a cartilage disease of the present invention is capable of demonstrating therapeutic effects on a wide range of injection cartilage trauma injuries to a joint in a liquid state. The composition demonstrates at least one effect selected from the group consisting of cartilage repair in a cartilage trauma injury as seen, for example, in aging, trauma, osteoarthritis, discus injury, meniscal injury or dissecting osteochondritis, 15 inhibition of changes degenerative cartilage, and cartilage protection.

Furthermore, the composition for treating a cartilage disease of the present invention has the effect of inhibiting joint inflammation and suppressing pain associated with inflammation. The composition demonstrates analgesic action by inhibiting inflammatory joint responses, for example in osteoarthritis, frozen shoulder and rheumatoid arthritis.

The cartilage disease treatment composition of the present invention is capable of inhibiting the progression of cartilage disease and alleviating or curing symptoms by demonstrating reparative, protective and inhibitory effects on mechanical cartilage damage while inhibiting , inflammatory responses and joint tissue pain. In particular, the composition is useful for treating osteoarthritis, treating frozen shoulder and relieving joint pain associated with rheumatoid arthritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing cell survival rates versus concentration of various CaCl2 solutions (Example 4).

FIG. 2 is a graph showing the comparative effects of alginate purified and food-grade blood cells on cell survival rates in cells (Example 5).

FIG. 3 is a graph showing the results of RT-PCR analyzes during in vitro cultivation of Example 6.

FIG. 4 shows photographs of the staining results during in vitro cultivation of Example 6. (A) 15 purified sodium alginate - 21 days of cultivation; (B) purified sodium alginate - 28 days of cultivation; (C) food grade sodium alginate - 21 days of cultivation; (D) food grade sodium alginate - 28 days of cultivation. Staining was performed from left to right, H-E staining, safranin-O staining, anti-type I, anti-type II, and anti-type X anti-collagen antibodies.

FIG. 5 shows photographs showing images taken during a procedure in a rabbit cartilage repair model of Example 7.

FIG. 6 is a graph showing the criteria for overall score observations in a rabbit cartilage repair model of Example 7.

FIG. 7 is a graph showing the criteria for

30 Score of staining results in a rabbit cartilage repair model of Example 7.

FIG. 8 shows tissue staining photographs of a control group (A) (empty) in a rabbit cartilage repair model of Example 7. FIG. 8A shows the 5 results after 4 weeks, while FIG. 8B shows results after 12 weeks. Results are shown for, from left to right, H-E staining, safranin-O staining, and type I collagen and type II collagen immunostaining.

FIG. 9 shows tissue staining photographs of

a food grade alginate + cells (group C) in a rabbit cartilage repair model of Example 7. FIG. 9A shows results after 4 weeks, while FIG. 9B shows results after 12 weeks. Staining methods are the same as those of FIG. 8

FIG. 10 shows tissue staining photographs of a purified (cell-free) alginate (group D) in a rabbit cartilage repair model of Example 7. FIG. IOA shows results after 4 weeks, while FIG. IOB 20 shows results after 12 weeks. Staining methods are the same as those of FIG. 8

FIG. 11 shows tissue staining photographs of a purified alginate + cells (group E) in a rabbit cartilage repair model of Example 7. FIG. IlA 25 shows the results after 4 weeks, while FIG. IlB shows results after 12 weeks. Staining methods are the same as those of FIG. 8

FIG. 12 shows the results of overall score and staining observations in a cartilage repair model

Rabbit 30 of Example 7. FIG. 13 is a graph showing the results of measuring mechanical strength for a purified alginate (groups D and E) in a rabbit cartilage repair model of Example 7.

FIG. 14 shows photographs taken during a

experiment on a corpse model from Example 8.

FIG. 15 is a graph showing the relationship between concentrations (%) and adhesion times (seconds) of various sodium alginate solutions.

FIG. 16 is a graph showing the relationship

between viscosity (mPa.s) and adhesion time (seconds) of a sodium alginate solution.

FIG. 17 shows photographs of the appearance of knee joints in a rabbit osteoarthritis model of Example 12.

FIG. 18 shows staining photographs of knee joint tissue tissue in a rabbit osteoarthritis model of Example 12.

FIG. 19 shows photographs of the appearance of knee joints in a rabbit osteoarthritis model of Example 13 following India ink staining. In the photographs, the circled areas indicate the boundaries between cartilage trauma injuries stained with ink and normal cartilage. A) Control group; B) 1% sodium hyaluronate dose group; C) 2% sodium alginate dose group (molecular weight: 400,000); D) 2% sodium alginate dose group (molecular weight: 1,000,000); E) 2% sodium alginate dose group (molecular weight: 1,700,000). In addition, the photographs show examples of various samples. FIG. 20 shows the results of macroscopic findings of ink-stained knee joints in a rabbit osteoarthritis model of Example 13. NS, HA, AL40, AL100 and AL170, respectively, correspond to (A) to (E) ( same as Figure 19). Grade 1 indicates an uninjured surface not stained with ink (no ink pickup indicating intact surface). Grade 2 indicates focal staining with nanjing ink and slight surface damage (minimal focal pickup of nanjing ink, slight surface irregularity). Grade 3 indicates large, well-defined staining with nankin ink and obvious fibrillation (large focal dark spots evident from nankin ink, frank fibrillation). Grade 4a indicates cartilage erosion less than 2 mm (cartilage erosion <2 mm). Grade 4b indicates cartilage erosion of

2 to 5 mm (cartilage erosion 2-5 mm). Grade 4c indicates cartilage erosion above 5 mm (cartilage erosion> 5 mm).

FIG. 21 shows photographs of the tissue staining of the

2 0 knee joint with safranin-O in a model of

Rabbit osteoarthritis of Example 13. (A) to (E) are the same as FIG. 19. In addition, the photographs show examples of various samples.

FIG. 22 shows the results of the overall histopathological evaluation score in a rabbit osteoarthritis model of Example 13. NS, HA, AL40, AL100 and AL170, respectively, correspond to (A) to (E) (same as FIG. 19).

FIG. 23 shows time-based changes in

30 Gait scores in an experimental rat model of arthritis pain from Example 15. A) Control group (NS); B) 1% sodium hyaluronate dose group (1% HA); C) 2% sodium alginate dose group (molecular weight: 1,000,000)

(AL100 2%); D) 1% sodium alginate dose group (molecular weight: 1,700,000) (1% AL170); E) 2% sodium alginate dose group (molecular weight: 1,700,000) (2% AL170). *: p <0.05 vs. NS

[FREE TEXT OF LISTING OF SEQUENCES]

ID SEQ NO 1: Synthetic DNA ID. SEQ No.2: Synthetic DNA ID. SEQ No.3: Synthetic DNA ID. SEQ No. 4: Synthetic DNA ID. SEQ No. 5: Synthetic DNA ID. SEQ No. 6: Synthetic DNA ID. SEQ No. 7: Synthetic DNA ID. SEQ No. 8: Synthetic DNA ID. SEQ No 9: Synthetic DNA ID. SEQ N0 10 Synthetic DNA BEST MODE FOR CARRYING OUT THE INVENTION

2 0 Although an explanation is given below

In the following detailed embodiments of the present invention, the following embodiments are exemplary for explaining the present invention, and the present invention may be embodied in various ways without departing from its scope.

1. Introduction

"Cartilage" is found in the joints; chest wall; intervertebral discs; meniscus; tubular structures such as the throat, respiratory tract and ears; and so on; and is

classified into three types consisting of hyaline cartilage, elastic cartilage and fibrous cartilage. For example, articular cartilage is classified as hyaline cartilage, is composed of chondrocytes, collagenous extracellular matrix, proteoglycan and water, and has no vascular intervention. Hyaline cartilage is rich in type II collagen, and is stained by type II collagen antibodies. It is also characterized by being stained by safranin-0 staining used for proteoglycan staining. "Cartilage trauma" refers to a state in which cartilage has been damaged as a result of aging, trauma, or other factors, and includes a state in which cartilage function has decreased with, for example, a decrease in characteristic viscoelasticity. cartilage (which allows the cartilage to slowly compress when loaded and then slowly return to its original state when the load is removed), thereby reducing the ability of the cartilage to withstand a load while maintaining mobility . Cartilage damage is observed in diseases such as osteoarthritis and rheumatoid arthritis. The present invention relates to a curable cartilage regeneration composition that can be applied to such a cartilage trauma injury. A "cartilage defect" refers to a cartilage trauma injury in which a portion of the cartilage is absent, and indicates a void in the cartilaginous tissue and surrounding tissue that forms the void. The composition of the present invention is preferably used for treating a "cartilage defect".

More specifically, the present invention is a

The cartilage regeneration composition that is applied to a cartilage trauma injury contains a monovalent metal salt of alginic acid for which the endotoxin level thereof has been reduced to a degree that does not substantially induce fever or inflammation, and has a viscosity of 400 to 20,000 mPa.s.

Accordingly, as the composition of the present invention is capable of effectively promoting cartilage regeneration in an affected area, it demonstrates satisfactory adherence to cartilage trauma injury and can be applied with a syringe or the like, is easily applied to a trauma injury. of cartilage.

In the present invention, "cartilage regeneration" or "cartilage tissue regeneration" refers to the restoration of the function of a cartilage trauma injury for which cartilage function has been impaired or lost. In the present invention, restoration of function does not necessarily require function to be completely restored, but rather that function to be restored to a greater degree than the state of cartilage trauma injury prior to application of the present composition. If a value of 100% is attributed to normal cartilage status prior to injury and a value of 0% to cartilage trauma status prior to application of the present composition, cartilage function is preferably restored to 30% or more thereof. more preferably up to 50% or more of this, even more preferably up to 80% or more of this and particularly preferably to a state almost equivalent to that prior to injury. The proportion of non-hyaline cartilage, for example fibrous cartilage, in regenerated cartilage is preferably low. In addition, "treatment of a cartilage trauma injury" or "treatment of a cartilage defect" refers to the relief or cure of its symptoms by cartilage regeneration in cartilage trauma injuries or cartilage defects observed in the aging, trauma, osteoarthritis, discus or meniscal injury, dissecting osteochondrosis, and the like.

In addition, "application to a cartilage trauma injury" refers to the use of a composition for cartilage regeneration and the like upon contact with a cartilage trauma injury and preferably to the use of the composition of the present invention by injection into a cartilage defect to fill that cartilage defect. Alternatively, the composition of the present invention may be used to further form one or more comparatively small holes in a cartilage trauma injury and preferably a cartilage defect, and injection of the composition of the present invention into the holes to fill the holes. The application to the injury by

The cartilage trauma is preferably performed in such a manner

so that the total void of an affected area is sufficiently filled. The affected area is preferably subjected to the necessary pretreatment and irrigated, if necessary, prior to application of the present composition. Irrigation of the affected area refers to the use of saline, for example, to remove unnecessary blood and other tissue components and the like where the composition of the present invention is applied. After irrigation, the affected area is preferably dried for

For example, by wiping any remaining unnecessary and similar liquid components, followed by applying the composition of the present invention.

In the present invention, "cartilage disease" refers to a disease that occurs because the cartilage, cartilaginous tissue, and / or articular tissue (e.g., the synovial membrane, joint capsule, or subchondral bone) has been injured. mechanical irritation or inflammatory response. "Treatment of cartilage disease" refers to the alleviation, amelioration and / or cure of various symptoms of tissue that has been injured by mechanical irritation or inflammatory response. For example, in cases of osteoarthritis, compound symptoms such as joint cartilage wear, cartilaginous tissue degeneration, synovial membrane inflammation, or pain associated with inflammation occur. On the other hand, in cases of frozen shoulder, symptoms consist primarily of inflammation of the synovial membrane and joint capsule, and associated pain, while cartilage wear and degeneration may not be observed. Although the

2 The mechanism of occurrence of rheumatoid arthritis is not

Fully understood, synovial and cartilaginous tissue are believed to be destroyed by inflammatory cytokines resulting from an autoimmune response. Thus, cartilage disease is a disease that presents with compound symptoms, and drugs are needed to treat these that have compound effects, including protection of cartilage against wear, inhibition of degenerative changes in cartilage as a result of wear or tear. inflammation, repair of injuries from trauma of

3 0 cartilage, and inhibition of inflammation and pain. The "composition containing an alginic acid lower endotoxin monovalent metal salt" of the present invention has the effects of protecting cartilage against mechanical irritation, inhibiting degenerative changes in cartilage caused by wear or inflammation, repair of traumatic injury. cartilage, and inhibition of inflammation and pain of the joint tissue. As a result, the composition is capable of inhibiting the progression of cartilage disease, and alleviating, ameliorating and / or curing the symptoms. In particular, the composition is useful for treating osteoarthritis, treating frozen shoulder and relieving joint pain associated with rheumatoid arthritis.

In addition, "injection into a joint" refers to the injection of a fluid composition that has fluidity, for example, into an articular cavity, synovial sac or peritendinal region. When used for the treatment of osteoarthritis, the composition is preferably injected into a joint cavity. In addition, although osteoarthritis can occur in various joints of the body, including

2 0 those of the knees, shoulders, ribs, lower back,

ankles, wrists and toes, the composition of the present invention may be applied to any such joint.

2. Monovalent alginic acid metal salt The "monovalent alginic acid metal salt" contained in

in the composition for cartilage regeneration or treatment of a cartilage disease of the present invention is a water soluble salt formed by ion exchange between a carboxylic acid hydrogen atom at the 6 position of the

Alginic acid is a monovalent metal ion such as Na + or K +. While specific examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, sodium alginate which can be purchased as a commercially available product is particularly preferable. A solution of a monovalent alginic acid metal salt forms a gel when mixed with a crosslinking agent.

The "alginic acid" used in the present invention is a high molecular weight biodegradable polysaccharide, which is a polymer obtained by linear polymerization of two types of uronic acids as D-manuronic acid (M) and L-gluronic acid (G ). More specifically, alginic acid is a block copolymer in which a D-manuronic acid homopolymer fraction (MM fraction), an L-gluronic acid homopolymer fraction (GG fraction) and a fraction in which D- manuronic acid and L-gluronic acid are randomly arranged (MG fraction), are bound arbitrarily. The composite ratio of D-manuronic acid to L-gluronic acid of alginic acid (M / G ratio)

20 varies mainly according to the type of algae or other organism that serves as its origin, is affected by the habitat and season of that organism, and varies over a wide range of a G-rich type having an M / G ratio around 0.4, up to a rich M type that has a

2 5 M / G ratio of about 5.

A monovalent alginic acid metal salt is a polysaccharide and, although it is difficult to precisely determine the molecular weight, it generally has an average molecular weight of 10,000 to 10,000,000 and preferably 50,000 to 3,000,000. As the cartilage regeneration effect on cartilage trauma injuries, and particularly the hyaline cartilage regeneration effect, decreases if the molecular weight is excessively low, the monovalent metal salt of alginic acid used in the present invention preferably has a weight. weight average molecular weight of 500,000 or more. In particular, sodium alginate having a weight average molecular weight of 500,000 or more has the unexpected effect of regenerating hyaline cartilage, even in the state that does not contain embedded cells, and is suitable for use as a cartilage regeneration composition. In addition, as the cartilage regeneration effect also advantageously contributes to the repair of cartilage trauma injuries in cartilage diseases, it is also suitable for use as a composition for the treatment of cartilage disease. Indeed, superior therapeutic effects were observed for high molecular weight alginic acid compared to low molecular weight alginic acid in a rabbit OA model. Sodium alginate having a weight average molecular weight of 1,000,000 and

1,700,000, as determined by gel filtration chromatography, demonstrated inhibitory effects of degenerative cartilage alteration, cartilage protective effects and superior cartilage repair effects, 25 compared to sodium alginate having a molecular weight of 410,000. In calculating the molecular weight of a polysaccharide by gel filtration chromatography, there is usually the potential for a measurement error of 10 to 20%. For example, a molecular weight of 400,000 may fluctuate in

3 0 a range of 320,000 to 480,000, a molecular weight of 500,000 may fluctuate in a range of 400,000 to 600,000, and a molecular weight of 1,000,000 may fluctuate in a range of

800,000 to 1,200,000. Thus, the preferred weight average molecular weight range of a monovalent metal salt of

alginic acid for which the effects on cartilage are particularly greater is at least 500,000 or more, more preferably 650,000 or more, and even more preferably 800,000 or more. In addition to production being difficult as problems such as

for example, viscosity being excessively high when preparing an aqueous solution, or decreasing solubility if the molecular weight is excessively high, the weight average molecular weight is preferably 5,000,000 or less and more preferably 3,0,000 or less.

As high molecular weight substances derived from

natural origin typically do not have a single molecular weight, but rather consist of an aggregate of molecules that have various molecular weights, molecular weight is measured as a weight distribution

20 The molecular having a certain range. A technique of

Typical measurement is gel filtration chromatography. Typical examples of information obtained from molecular weight distribution, as determined by gel filtration chromatography, include average weight molecular weight.

(Mw), average molecular weight (Mn) and ratio of variance (Mw / Mn).

The average weight molecular weight emphasizes the contribution of the average molecular weight of polymers having a large molecular weight, and is represented with the

3 0 following formula: Mw = Σ (WiMi) / W = Σ (HiMi) / Σ (Hi)

The average molecular weight is calculated by dividing the total polymer weight by the total number of polymers.

Mn = W / ΣΝϊ = Σ (MiNi) / Σνϊ = Σ (Hi) / Σ (Hi / Mi)

Here, W represents the total weight of all polymers,

Wi represents the weight of the ith polymer, Mi represents the molecular weight at the elution time of ith, Ni represents the number of molecular weights Mi, and Hi represents the height at the elution time of ith.

How the effects of cartilage regeneration (and

particularly the effects of hyaline cartilage regeneration) on cartilage trauma injuries, the effects of cartilage repair, the inhibition effects of degenerative cartilage changes and / or the protective effects of

Cartilage in the treatment of cartilage disease are considered to be greatly influenced by molecular species having large molecular weights, the average weight molecular weight can be used as an indicator of molecular weight.

2 0 Differences in values are known to occur according to

with methods for measuring molecular weights of high molecular weight substances derived from a natural origin (example of hyaluronic acid: Chikako Yomota et al., Bull. Natl. Health Sci., Vol. 117, pages 135-139

(1999), Chikako Yomota et al., Bull. Natl. Health Sci., Vol. 121, pages 30-33 (2003)). The methods for measuring alginate molecular weight described in the literature include a method in which the molecular weight is calculated from intrinsic viscosity, and a method in which the

30 Molecular weight is calculated by size exclusion chromatography with Multi-Angle Laser Light Scattering Detection (SEC-MALLS) (ASTM F2064-00 (2006), published by ASTM International). In addition, it is also described in the literature that, when measuring molecular weight by size exclusion chromatography (gel filtration chromatography), the calculation from a calibration curve using pullulan for the standard substance is insufficient, and recommends The molecular weight measurement is intended to be used in combination with the Multi-Angle Laser Light Scattering Detector (MALLS) (specifically, SEC-MALLS measurement). In addition, there are also examples of the use of SEC-MALLS-determined molecular weights that are used as alginate catalog specifications (FMC Biopolimer Inc., "PR0N0VA ™ Sodium Alginates Catalog").

The inventors of the present invention found that there are differences in the therapeutic effects of sodium alginate with different molecular weights in an OA model, and measured the molecular weights of these alginates by gel filtration chromatography and SEC-MALLS. As a result, molecular weights were determined by gel filtration chromatography to demonstrate a greater correlation with the viscosity and therapeutic effects of alginates. Specifically, it has now been found that, instead of the generally recommended SEC-MALLS method, the molecular weight determined by gel filtration chromatography is suitable as a parameter for specifying the preferred molecular weight range of alginates used in a composition for regeneration of

30 Cartilage or treatment of cartilage disease. Thus, when specifying the molecular weight of an alginate in the present specification, that molecular weight is the weight average molecular weight calculated by gel filtration chromatography unless specifically stated otherwise.

Preferred conditions for gel filtration chromatography are as indicated in the examples. A typical condition is the use of a calibration curve using pullulan for the standard substance. Preferably it is

pullulan with a molecular weight of at least

1,600,000, 788,000, 404,000, 212,000 and 112,000 for the standard substance. In addition, the eluate (200 mM sodium nitrate solution), column conditions and the like can also be specified. The column conditions

preferably consist of the use of a polymethacrylate resin-based filler and using at least one column with a molecular weight cut-off value of

10,000,000 or more. A typical example of a column is the TSkgel GMPWxl column (diameter: 7.8 mm x 300 mm) (Tosoh

20 Corp.).

Although a monovalent alginic acid metal salt has a large molecular weight and high viscosity when initially isolated from brown algae, the molecular weight decreases and the viscosity decreases during the evolution of the algae.

heat drying, lyophilization, purification, and the like. Thus, monovalent metal salts of alginic acid with different molecular weights can be produced by properly controlling the temperature in each production step. Salts

30 Monovalent alginic acid metals having a high molecular weight are obtained by controlling the temperature at each of the production steps to be somewhat low, while monovalent metal salts of alginic acid having a low molecular weight 5 are obtained. by controlling the temperature in each production step so that it is a little high. In addition, monovalent metal salts of alginic acid of different molecular weights can also be produced by a technique such as, for example, by appropriately selecting the 10 brown algae used as raw material, or by fractionating according to the molecular weight in the sample. production process. In addition, a monovalent alginic acid metal salt of a target molecular weight may also be obtained by mixing a monovalent alginic acid metal salt 15 produced according to various production processes with a different batch of the monovalent metal salt. of alginic acid of a different molecular weight or viscosity after its molecular weight or viscosity has been measured.

Although the alginic acid used in the present invention

may be of natural or synthetic origin, it is preferably derived from a natural origin. Examples of naturally occurring alginic acids include those extracted from brown algae. Although brown algae containing 25 alginic acid are found predominantly along the coasts around the world, algae that can actually be used as alginic acid feedstocks are limited, with typical examples of these including Lessonia species found in South America. , species of

3 0 Macrocystis found in North America, Laminaria and Ascophyllum species found in Europe and Durvillea species found in Australia. Examples of brown algae that serve as alginic acid feedstocks include Lessonia species, Macrocystis species, 5 Laminaria species, Ascophyllum species, Durvillea species, Eisenia species, and Ecklonia species.

3. Endotoxin Reduction Treatment

The monovalent metal salt of alginic acid contained in the cartilage regeneration composition or treatment of a cartilage disease of the present invention is a lower endotoxin monovalent metal salt of alginic acid. Lower endotoxin refers to the fact that its endotoxin level has been substantially reduced to a degree that does not induce inflammation or fever. Specifically, the monovalent metal salt of alginic acid was subjected to endotoxin reduction treatment. It has been surprisingly found that by being subjected to this endotoxin reduction treatment, in addition to being able to increase the regenerative action of the cartilage of the composition when

20 applied to a cartilage trauma injury, subchondral bone regeneration may be promoted and the mechanical strength of the affected area may be increased. Specifically, by using lower endotoxin alginic acid in the composition of the present invention, a composition having high bioaffinity can be obtained which does not induce degeneration and inflammatory responses in the surrounding cartilage.

Endotoxin reduction treatment may be performed by a known method or by a compatible method. For example, this treatment can be performed by the method of Suga et al. involving the purification of sodium hyaluronate (see, for example, Japanese Open Patent Application No. H9-324001) by the method of Yoshida et al. involving the purification of β, 3-glucan (see, for example, Japanese Open Patent Application No. H8-269102) by the method of William et al. which involves the purification of a biopolymer such as alginate or gellan gum (see, for example, PCT International Publication No. 2002-530440) by the method of James et al. involving the purification of polysaccharide (see, for example, International Publication Pamphlet No. 93/13136) by the method of Lewis et al. (see, for example, U.S. Patent No. 5,558,591) by the method of Hermanfranck et al. involving the purification of alginate (see, for example, Appl. Microbiol. Biotechnol. (1994), 40: 638-643), or by a compatible method. The endotoxin reduction treatment of the present invention is not limited to such methods but may be performed by a method known as, for example, cleaning, purification using filter filtration (endotoxin removal filter or electrification filter). ), ultrafiltration or a column (for example, an endotoxin adsorption affinity column, gel filtration column or ion exchange column), adsorption to a hydrophobic substance, resin or activated carbon, and the like, organic solvent treatment ( for example extraction with an organic solvent or precipitation or deposition by addition of organic solvent), surfactant treatment (see, for example, Japanese Open Patent Application No. 2005-036036), or a suitable combination thereof. A method known as, for example, centrifugal separation may be suitably combined with such treatment steps. Preferably, the endotoxin reduction treatment is suitably selected according to alginic acid type 5.

The endotoxin level can be confirmed by a known method, and can be measured using a known method such as, for example, a method using a limulus reagent (LAL) or a method using a 10 Endospecy (trademark) ES- 24S (Seikagaku Corp.). Although there are no specific limitations on the method of treating alginic acid endotoxin contained in the composition of the present invention, the endotoxin content of the monovalent alginic acid metal salt, when measuring endotoxin using a limulus reagent (LAL), is preferably from 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, even more preferably 50 EU / g or less and in particular preferably 30 EU / g or less as a result thereof.

2 0 method. Sodium alginate that has undergone endotoxin reduction treatment can be purchased as commercially available products such as "Sea Matrix" (sterile) (Kimica Corp., Mochida International Ltd.) and Pronova ™ UP LVG (FMC).

2 5 4. Preparation of a metal salt solution

monovalent alginic acid

The composition for cartilage regeneration or treatment of cartilage disease may be prepared by using a solution of a monovalent alginic acid metal salt. The solution of a monovalent metal salt of alginic acid may be prepared by a known method or by a compatible method. Specifically, the monovalent alginic acid metal salt used in the present invention may be produced by a method known as, for example, an acid method or a calcium method using the brown algae described above. More specifically, after extraction of such brown algae using an alkaline aqueous solution such as an aqueous sodium carbonate solution, for example, alginic acid may be obtained by the addition of an acid (eg hydrochloric acid or sulfuric acid ), and an alginic acid salt can be obtained by ion exchange of the alginic acid. Endotoxin reduction treatment is then performed as previously described. There are no specific limitations on the alginic acid salt solvent as long as it is a solvent that can be applied in vivo, and examples of such solvents include purified water, distilled water, ion exchange water, Milli-Q water, saline, and phosphate buffered saline (PBS). These are preferably sterile and preferably subjected to endotoxin reduction treatment. For example, Milli-Q water may be used after filter sterilization. The cartilage regeneration or cartilage disease treatment composition of the present invention may also be obtained, for example, by mixing a monovalent alginic acid metal salt in a cell-containing medium without dissolving the metal salt. monovalent solution of alginic acid in the solvent mentioned above. In addition, the procedure for obtaining the composition of this

The invention is preferably carried out in an environment with low levels of endotoxins and bacteria. For example, the procedure is preferably performed on a clean bench using sterile apparatus, and used apparatus may be treated with a commercially available endotoxin removal agent.

When producing a composition as described above using a monovalent alginic acid metal salt that has been purified to a preferable endotoxin level, the endotoxin content of the composition is

usually 500 EU / g or less, more preferably 300 EU / g or less and especially preferably 150 EU / g or less.

5. Viscosity of the composition for cartilage regeneration or for treatment of cartilage disease

Although there are no specific limitations on the

viscosity of the cartilage regeneration composition of the present invention provided that the effects of the present invention are obtained, it is preferably from 400 to 20,000 mPa.s. The composition of the present invention may be adjusted to

20 to a suitable viscosity per use, e.g.

of the solvent mentioned above. If the viscosity is within this range, adherence to a cartilage trauma injury is favorable and the composition may be injected into a joint cavity or cartilage trauma injury.

with a syringe or the like. In addition, if the viscosity of the cartilage regeneration composition is about 2,000 mPa.s or more, adherence to a cartilage trauma injury will be further enhanced, and if the viscosity is about 5,000 mPa.s or more

30, in particular, even if the opening of a cartilage defect is turned downward, for example in the case of arthroscopic manipulation of cartilage trauma on the surface of a human femoral joint, for example, the composition of the present invention may be brought into contact with the surface of the injection cartilage trauma injury of the composition of the present invention in the cartilage defect and allowed to adhere therein for at least one minute in the absence of fixation. The surface of the composition may be fixed, if necessary, for as long as it is adhering. Adherence to cartilage trauma injury increases further as viscosity increases and, in case of

10,000 mPa.s, for example, the composition may be adhered without fixation for a longer period of time compared to a viscosity of 5,000 mPa.s. Thus, when the composition of the present invention is applied to a cartilage trauma injury in the state in which an opening of a cartilage defect or an opening of a hole formed in a cartilage trauma injury or cartilage defect is slanted. or face down, the composition of the present invention will be adhered to the cartilage trauma injury for at least 5 seconds, preferably for at least 10 seconds, more preferably for at least 30 seconds, and especially for at least 1 second. minute without the use of a fastening means. As a result of adjusting its viscosity, the composition of the present invention may remain long enough until a fixing medium is applied to the surface of the composition. Here, "grip

30 to a Cartilage Trauma Injury "refers to the permanence of the composition of the present invention in the cartilage trauma injury without leaving it. Thus, as a result of adjusting its viscosity, the composition of the present invention offers the advantage It should be possible for the treatment to be performed by a simple injection procedure, even if the affected area is positioned in such a way that it is difficult for a surgeon to perform the treatment, for example when the affected area is turned down.

On the other hand, the composition of the present invention is

injected more easily with a syringe or the like when its viscosity is about 20,000 mPa.s or less. Although the composition may be injected with a syringe or the like even if, for example, its viscosity is about 20,000 mPa.s, when injection is difficult due to excessively high viscosity, the composition of the present invention may be applied to the composition. surface of a cartilage trauma injury using other means. From the point of view of ease of handling the syringe, the viscosity of the composition of the present invention is preferably 20,000 mPa.s or less and more preferably 15,000 mPa.s or less. Accordingly, the viscosity of the composition of the present invention applied to a cartilage trauma injury in the state in which an opening of a cartilage defect or an opening of a hole formed in a cartilage trauma injury or cartilage defect is slanted. or facing down is preferably about 2,000 mPa.s or more from the point of view of adhesion, preferably

30 about 20,000 mPa.s or less from the point of view of ease of manipulation of the composition and preferably

3,000 to 15,000 mPa.s, more preferably 4,000 to 10,000 mPa.s, and particularly preferably 5,000 to 6,000 mPa.

If the viscosity of the composition of the present invention is about 400 mPa.s or more, the composition will be able to adequately demonstrate the effects of the present invention when applied to a cartilage trauma injury. For example, when the present invention is capable of operating in the state in which the opening-containing side of a cartilage defect is facing upward, the composition of the present invention may be injected into the cartilage trauma injury so that there is contact with it. of the composition of the present invention with the surface of the cartilage trauma injury, followed by fixation of the surface of the composition. Injection with a syringe or the like can be easily performed due to the low viscosity of the composition. If the viscosity of the composition is about 5000 mPa.s, for example, the composition may be applied to the entire cartilage trauma injury by forming one or more extremely small holes in the cartilage trauma injury such as when there is residual cartilage at the site of cartilage trauma injury.

While there are no specific limitations on the injection viscosity of the composition for treating a cartilage disease of the present invention in a joint provided that the therapeutic effects on cartilage disease are obtained, the viscosity is preferably from 100 to 20,000 mPa. s. The viscosity is preferably 200 to 15,000 mPa.s, more preferably 400 to 10,000 mPa.s, and particularly preferably 1,000 to 6,000 mPa.s. The use of adequate viscosity makes it possible to demonstrate the compensatory effect of the damping function of synovial fluid, thereby making it possible to demonstrate the effect of treating a cartilage disease in a dispersed state in the synovial fluid.

The viscosity of the composition for cartilage regeneration or treatment of a cartilage disease may be adjusted, for example, by controlling the alginic acid concentration in the solution of a monovalent alginic acid metal salt or by controlling the molecular weight. of alginic acid.

The viscosity of the alginic acid monovalent metal salt solution increases when the alginic acid concentration in the solution is high and decreases when the alginic acid concentration in the solution is low. Although it is not possible to unambiguously establish the influence of molecular weight, the preferable concentration of alginic acid in the monovalent metal ion solution of

Alginic acid is approximately 1 to 5% w / v, more preferably 1.5 to 3% w / v and especially preferably 2 to 2.5% w / v.

The monovalent alginic acid metal salt initially has a high molecular weight and viscosity.

2 5 increased when extracted from brown algae, although the

concentration is constant, and molecular weight decreases during the process of heating drying, lyophilization, purification and the like, eventually resulting in somewhat low viscosity. The viscosity

30 varies constantly for alginic acid extracted from the same brown algae. In addition, the measured viscosity values also vary according to the measuring instrument and the measuring conditions. Thus, a solution of a monovalent alginic acid metal salt 5 which is more adherent to a cartilage trauma injury and for which its endotoxin level has been reduced is included within the scope of the present invention.

A monovalent alginic acid metal salt having a high molecular weight may be selected to obtain a composition with a tack greater than an affected area and high viscosity by a solution of a low concentration monovalent alginic acid metal salt.

As the solution viscosity of a monovalent alginic acid metal salt is affected by the M / G ratio, an alginic acid having a more preferable M / G ratio for solution viscosity and the like may be suitably selected. The M / G ratio of alginic acid used in the present invention is about 0.4 to 4.0, preferably about 0.8 to 3.0, and more preferably about 1.0 to 1.6.

As previously described, as the M / G ratio is determined primarily by the type of algae, the type of brown algae used for the raw material has an effect on the viscosity of the monovalent alginic acid metal salt solution. The alginic acid used in the present invention is preferably derived from brown algae of the genera Lessonia, Macrocystis, Laminaria, Ascophyllum and Durvillea, more preferably derived from brown algae.

30 of the Lessonia genera and particularly preferably brown algae of Lessonia nigrescens.

In addition, the viscosity of the composition may be adjusted, for example, by the amount of embedded cells (see description below) present in the solution of the monovalent alginic acid metal salt. If the composition of the present invention contains embedded cells, the viscosity of the composition of the present invention will preferably be adjusted based on the viscosity of the composition after the cells have been embedded. However, when using embedded cells in clinical practice, it is difficult to perform a viscosity adjustment step after the cells have been embedded. Thus, when the composition of the present invention has embedded cells, the viscosity of the composition before the cells are embedded may be considered to be the viscosity of the composition of the present invention.

One aspect of the composition of the present invention is a composition for cartilage regeneration by application to a cartilage trauma injury wherein bone marrow mesenchymal stem cells and / or bone marrow mesenchymal stromal cells are included in a composition containing a monovalent alginic acid metal salt for which the endotoxin level thereof has been reduced to a degree that does not substantially induce fever or inflammation, has a viscosity of 400 to 20,000 mPa.s, and has fluidity.

6. Soaked Cells

The composition for cartilage regeneration or treatment of a cartilage disease of the present invention.

The invention may have cells embedded for the regeneration of cartilage tissue in a composition containing a monovalent metal salt of alginic acid and preferably may have cells embedded for the regeneration of cartilage tissue in a solution of a monovalent metal salt of alginic acid. alginic acid. The term "soaked" as used herein refers to the cell suspension for regeneration of cartilaginous tissue in a composition containing a monovalent metal salt of alginic acid and preferably to cell suspension for the generation of cartilaginous tissue in a solution of a monovalent metal salt of alginic acid. As a result, cartilage regeneration may be promoted more effectively and the cartilage resistance to which the composition of the present invention has been applied may be further increased. Preferably, the cells are dispersed in the composition of the present invention. While examples of these cells include stem cells and stromal cells, and there are no specific limitations on their origin, examples of these include bone marrow cells, adipocytes, and umbilical cord blood. Such cells are preferably bone marrow mesenchymal stem cells or bone marrow mesenchymal stromal cells. Other examples include cartilage precursor cells, chondrocytes, synoviocytes, erythropoietic stem cells and ES cells. One or more of these cells may be embedded. Because "stem cells" in particular have self-regeneration and multiple differentiation ability, the use of these stem cells for cartilage regeneration allows for histologically superior cartilage regeneration with superior mechanical strength. Although stem cells include embryonic stem cells and bone marrow mesenchymal stem cells, as bone marrow mesenchymal stem cells allow the use of adult autologous cells, they are easily acquired and are suitable for use for cartilage regeneration. . In addition, because bone marrow mesenchymal stem cells can differentiate into both bone and cartilage, in cases where, for example, an injury extends to the bone, in addition to cartilage, these cells are able to regenerate bone. at the bone site and cartilage at the cartilage site. By suspending bone marrow mesenchymal stem cells in a solution of a monovalent alginic acid metal salt and injecting it into a joint, the suspension can be used to treat cartilage disease. Thus, although the cells used in the present invention preferably consist of a high proportion of bone marrow mesenchymal stem cells, as it is difficult to isolate these cells completely, bone marrow mesenchymal stem cells are among the most common.

20 cells for the regeneration of cartilaginous tissue of the present invention preferably at 30% or more, more preferably at 50% or more, even more preferably at 70% or more and especially preferably at 90% or more. One aspect of the composition of the present invention is a composition in which bone marrow mesenchymal stem cells and / or bone marrow mesenchymal stromal cells are used for cartilage regeneration or for treatment of cartilage disease.

Although the embedded cells may be heterologous cells or autologous cells, autologous cells are preferably collected and used from the standpoint of avoiding particular rejection reactions. The collected cells are preferably used after cell culture proliferation. At this time, the cells may initially be soaked in a solution of a monovalent alginic acid metal salt and then cultured while in that state, or the cells may be soaked in a solution of a monovalent alginic acid metal salt after cultivation. of cells in a culture medium. 10 Cells may be cultured according to a

common method, and cells may be cultured while soaked in a solution of a monovalent alginic acid metal salt, or may be cultured without being soaked in a solution of a monovalent alginic acid metal salt. A medium that allows the cultivation of cells soaked in a solution of a monovalent metal salt of alginic acid as well as cells not soaked in this solution to be performed efficiently is preferable to the culture medium, and the culture medium may be selected

20 suitably by those skilled in the art from known means.

Examples of media that may be used include DMEM medium (Virology, Vol. 8, 396 (1959)), MEM medium (Science, Vol. 122, 501 (1952)), RPMI1640 medium (The Journal of the 25 American Medical Association). , 199, 519 (1967)) and F12, and serum, amino acids, glucose or antibiotics and the like may be added if necessary. The pH is preferably from about 6 to 8. Cultivation is usually carried out at about 30 to 40 ° C for 5 to 120 hours and preferably for 5 to 100 hours. In addition, the media may be changed, aerated or agitated if necessary.

In one aspect of the present invention, the composition contains a solution of a monovalent alginic acid metal salt mixed with cells for the regeneration of cartilaginous tissue and particularly stem cells.

mesenchymal bone marrow or bone marrow mesenchymal stromal cells, and does not contain growth factor such as TGF-β. Moreover, cell differentiation need not necessarily be induced in vitro. In this case, if fluid from the anterior margin of the iliac marrow is collected from a patient with cartilage trauma, for example, the bone marrow mesenchymal stem cells are immediately removed from the bone marrow fluid and the number of cells obtained 15 from it. region is a certain number of cells or more, the cells may be applied to the patient directly in the form of the composition of the present invention. As there is no concern to cultivate and differentiate in vitro collected cells, the procedure is extremely advantageous for the surgeon, costs can be reduced, and the burden on the patient can be attenuated.

In addition, bone marrow mesenchymal stem cells have superior practicability in allowing allogeneic cells to be applied without incident due to their low immunogenicity.

On the other hand, a solution of a monovalent alginic acid metal salt embedded with the cells described above may be used as a composition, wherein the embedded cells are cultured in vitro prior to

Application to a cartilage trauma injury in any condition selected from the group consisting of: a) a state in which the number of cells is 1 x 106 cells / mL or more, b) a state in which similar cartilage tissue hyaline is detected by safranin-0 or 5 HE staining, c) a state in which type II collagen is detected by anti-collagen II antibody or genetic analysis, d) a state in which agglican is detected by anti-agglutic antibody or genetic analysis, ee) a state in which the extracellular matrix (collagen, hyaluronic acid, proteoglycan) is secreted. The condition may be appropriately selected according to the state of the injury and the condition of the patient.

While there are no specific limitations on the amount of cells embedded, it can be, for example, 1.0 x 10 7 to 3.0 x 10 7 cells / ml and preferably 2.0 x 10 7 to 3.0 x 10 7 cells. / ml. Cartilage regeneration can be promoted most effectively by utilizing this number of cells.

On the other hand, it is preferable to use a cell-free composition 20 to facilitate the surgical procedure, as well as to reduce the risk of infection by viruses and the like attributable to the body or the cultivation process without placing an excessive burden on the body. by procedures such as chondrocyte collection, periosteum or 25 bone marrow. A preferred example of such a composition is a cartilage regeneration composition having a viscosity of 400 to 20,000 mPa.s and which has healing fluidity in an area affected by application to a cartilage trauma injury comprising a composition without

30 cells containing lower endotoxin sodium alginate having a weight average molecular weight as determined by silica gel filtration chromatography.

500,000 or more. In addition, the composition for treating a cartilage disease of the present invention is a composition having as an active ingredient a monovalent metal salt of lower endotoxin of alginic acid, and is based on the finding that alginic acid itself has a therapeutic effect on cartilage disease. An example of a preferred therapeutic composition is a composition for treating a cartilage disease that is injected into a joint, which comprises a cell-free composition which contains as an active ingredient this lower endotoxin sodium alginate having a molecular weight. weight ratio as determined by gel filtration chromatography of 500,000 or more, and is capable of demonstrating therapeutic effects that are superior to those of hyaluronic acid preparations used in the established technique.

7. Gelling the surface of the composition In the present invention, a composition containing a

A solution of a monovalent alginic acid metal salt may be applied to a cartilage trauma injury, and a crosslinking agent may be applied to the surface of the composition. Gelling the surface of composition 25 to solidify the surface makes it possible to effectively prevent extravasation of cartilage trauma injury composition.

There are no specific limitations on the crisscrossing agent as long as it is able to solidify

30 a surface of a solution of a monovalent alginic acid metal salt by crisscrossing that solution, and examples include divalent or more metal ion compounds such as Ca2 +, Mg2 +, Ba2 + or Sr2 +, and crisscrossing reagents. have 2 to 4 amino groups in one molecule of this. Specific examples of divalent metal ion compounds or more include CaCl 2, MgCl 2, CaSO 4 and BaCl 2, while specific examples of crosslinking reagents having 2 to 4 amino groups in such a molecule include diaminoalkanes which optionally have a lysyl group (-CoCH (NH2) - (CH2) 4-NH2) on a nitrogen atom, specifically derivatives that form lysylamino groups as a result of replacing a diaminoalkane and amino group with a lysyl group. While specific examples of these include diaminoethane, diaminopropane and N- (lysyl) diaminoethane, a CaCl2 solution is particularly preferable for reasons such as ease of acquisition and gel strength.

There are no specific limitations on the method used to add a divalent or more metal ion to the surface of the composition, and an example of such a method is to apply a solution of a divalent or more metal ion to the surface of the composition with a syringe or vaporizer. The crosslinking agent may be applied to the surface of the present composition.

2 5 invention after or at the same time as the application of the

composition of the present invention in a cartilage defect.

The amount of crosslinking agent applied is preferably adjusted accordingly according to the

30 The size of the defect where the composition of the present invention is applied. The crosslinking agent gradually penetrates into the surface of the composition to which it is applied, and then the crosslinking progresses. The amount of the crosslinking agent applied is adjusted so as not to be excessive to prevent the crosslinking agent from having an excessively strong effect on the spot where the composition of the present invention comes into contact with a cartilage trauma injury. There are no specific limitations on the amount of divalent or more metal ion applied as long as it is capable of solidifying the surface of the composition containing a monovalent metal salt of alginic acid. However, when adding a 100 mM CaCl2 solution, for example, the amount added is preferably about 0.3 to 15.6 ml in the case of a defect having a diameter of about 5 mm. , and a depth of about 2 mm, and the dosage may also be determined in proportion to the surface area of the affected part. For example, the amount added is preferably from about 1 to 12 ml and more preferably from about 2 to 10 ml, in the case of a defect having a width (10 mm x 20 mm) and depth of about 5 mm. The amount added may be adjusted accordingly by observing the state of the cartilage trauma injury. The crosslinking agent 25 may be applied, for example, by continuous and slow application to the surface of the composition containing a monovalent alginic acid metal salt of ten to several seconds or more.

Furthermore, by the presence in the composition of the present invention of a crosslinking agent by which gelation is promoted by environmental changes such as a time difference, temperature difference or contact with calcium ion within the body, it can be A composition is maintained that maintains a liquid state prior to administration and gels spontaneously after administration to the body. Examples of such crosslinking agents include calcium glyconate, CaSO4 and calcium alginate.

Here, when calcium comes into contact with the crosslinking agent, it is known that a higher calcium concentration results in rapid gelation and formation of a more rigid gel. However, to the extent that calcium is cytotoxic, if its concentration is excessively high, there is a risk of having a detrimental effect on the regenerative action of the composition cartilage for cartilage regeneration or for the treatment of a cartilage disease. present invention. Therefore, when a CaCl2 solution is used to solidify the surface of a composition containing a monovalent alginic acid metal salt, for example, the calcium concentration is preferably 25 to 200 mM and more preferably 50 to 100 mM. .

Here, alginate globules are produced, for example, by dripping a sodium alginate solution into a CaCl2 solution followed by gelation. The 25 cell-soaked alginate globules are known to be used for cartilage regeneration (see, for example, References 2 and 3). However, although alginate globules need to be pressure-applied to a cartilage defect, as it is necessary to produce globules that match the size of the defect, their use in clinical practice is technically difficult. In addition, when a CaCl2 solution is used as a crosslinking agent, as Ca ions on the surface of the globules contact the surface of the cartilage trauma injury, there is also a cytotoxicity problem caused by calcium. In contrast, as the composition of the present invention is in the form of a solution, it can easily be applied to a defect of any shape, and any cartilage trauma injury 10 is capable of being coated with the composition, and adherence to the defect. cartilage is satisfactory. Since calcium concentration at the site where the composition comes in contact with the surface of the cartilage trauma injury can be kept at a low level, there are few problems with calcium cytotoxicity. To the extent that the effect of the crosslinking agent on the surface of the cartilage trauma injury contacted by the composition of the present invention is small, the composition of the present invention is able to easily contact

20 cells and tissue at cartilage trauma sites in the

body. About four weeks after application to a cartilage trauma injury, the composition of the present invention fuses with the tissue to a degree to which it is indistinguishable at the applied site, thereby demonstrating high bioaffinity.

When applying the composition of the present invention to a cartilage trauma injury, if applied first by mixing with a crosslinking agent to gel the entire composition with the

30 Cross-linking, or by placing a cross-linking agent on the surface of the composition, the composition of the present invention may be cured in the affected area and located therein to be adhered to the cartilage trauma injury where applied. As a result, when cells and the like have been embedded, components such as cells may be located in the affected area. Furthermore, as a result of the adhesion of the composition of the present invention to a cartilage trauma injury, the cartilage regeneration effects of the composition of the present invention and particularly the hyaline cartilage regeneration effects can be more powerfully demonstrated. .

8. Formulation and application of a cartilage regeneration composition or treatment of a cartilage disease containing a monovalent metal salt of alginic acid.

The composition for cartilage regeneration or treatment of a cartilage disease of the present invention is applied to a cartilage trauma injury 20 of a human or non-human mammal, such as a cow, monkey, bird, A cat, mouse, rat, guinea pig, hamster, pig, dog, rabbit, sheep, or horse is used to promote cartilage regeneration or treat injection cartilage disease. in a joint.

The form of the composition for cartilage regeneration or treatment of a cartilage disease of the present invention is preferably a fluid fluid, specifically a solution. In the present invention, the phrase "having fluidity" refers to the fact that it has a property that causes its shape to change to an amorphous shape, and does not require that the shape constantly have the fluidity property in the form of a shape. liquid, for example. For example, composition 5 preferably has a flowability such that it is capable of being sealed in a syringe or the like and injected into an affected area. The composition of the present invention in the form of a solution can be easily applied to a cartilage trauma injury or to a joint with a syringe, gel pipette or a specific syringe. In addition, it is compatible with any shape of cartilage trauma injury or defect, and is capable of filling in or contacting any cartilage defect.

The cartilage regeneration composition of the present invention demonstrates superior cartilage regenerative action in, for example, a hyaline cartilage cartilage defect in the form of articular cartilage. In addition, the composition for treating a cartilage disease of the present invention demonstrates adverse effects.

20 therapies in a cartilage disease such as

osteoarthritis, for having cartilage repair effects, effects that inhibit degenerative cartilage changes, protective effects of cartilage, effects that inhibit joint tissue inflammation and / or pain-suppressing effects attributable to joint tissue inflammation.

One aspect of the cartilage regeneration composition of the present invention is a composition for hyaline cartilage regeneration. One objective of a composition for the regeneration of hyaline cartilage is to

30 The cartilage regeneration with a high proportion of hyaline cartilage compared to fibrous cartilage aims to regenerate collagen-rich cartilaginous tissue type II and proteoglycan.

In addition, one aspect of the composition for treating a cartilage disease of the present invention is a composition for treating osteoarthritis. If a cartilage trauma extends over a wide area of articular cartilage in the form of osteoarthritis, or when a type of cartilage trauma 10 is often observed at a comparatively early stage of osteoarthritis such that the uniformity of the cartilage surface altered and degenerative changes have already begun, although well-defined cartilage defects have not yet occurred, the composition of the present invention will preferably be applied by injection into a joint cavity and allowed to disperse throughout the synovial fluid. Contact of a monovalent alginic acid metal salt with a cartilage trauma injury promotes

2 Joint repair in cartilage trauma injury, inhibits degenerative changes caused by inflammation and wear, and protects cartilage. In addition, because the active ingredient in the form of a monovalent alginic acid metal salt is dispersed throughout the synovial fluid, the inflammatory responses of surrounding tissue, including synovial tissue, are inhibited and pain-suppressing effects are impaired. demonstrated. At the same time, the presence of a monovalent alginic acid metal salt within the synovial fluid plays the role of compensating for the function of the synovial fluid by serving as a buffer and lubricant.

Another aspect of the composition for treating a cartilage disease of the present invention is a composition for treating frozen shoulder (scapular humerus periarthritis). Frozen shoulder presents primarily with inflammation of the synovial membrane and joint capsule coupled with the associated pain, and cartilage wear and degeneration may not be observed. As a monovalent alginic acid metal salt demonstrates the effects of

By inhibiting inflammatory responses of surrounding tissue, including synovial tissue, and suppressing pain, the frozen shoulder should be treated by administering the composition of the present invention to the shoulder joint cavity, subacromial pocket or biceps tendon sheath.

Another aspect of the composition for treating a

Cartilage disease of the present invention is a composition for suppressing joint pain. Joint pain is often a problem in rheumatoid arthritis, as well as osteoarthritis, frozen shoulder, and the like,

20 as previously described. A preferable aspect of

The present invention is a composition for the treatment of joint pain associated with rheumatoid arthritis, and a composition for the suppression of knee joint pain associated with rheumatoid arthritis is particularly preferred.

chronic. Although the mechanism of occurrence of rheumatoid arthritis is not yet fully understood, it is believed that synovial tissue and cartilaginous tissue are destroyed by inflammatory cytokines resulting from autoimmune responses. As a monovalent metal salt of

While alginic acid demonstrates effects that inhibit inflammatory responses of the surrounding tissue, including synovial tissue, and suppresses pain, the composition of the present invention is capable of inhibiting inflammatory responses and suppressing the pain associated with them by administration to a joint suffering from pain. rheumatoid arthritis. On the other hand, it is also necessary to suppress an autoimmune response in order to fundamentally treat rheumatoid arthritis, and it remains to be determined whether or not a monovalent alginic acid metal salt has immunosuppressive action in an area affected by rheumatoid arthritis.

Another aspect of the cartilage disease treatment composition of the present invention is a composition for alleviating, ameliorating and / or curing various symptoms associated with a cartilage disease. In a cartilage disease, cartilage, cartilaginous tissue and / or joint tissue (eg, synovial membrane, joint capsule or subchondral bone) are injured by mechanical irritation or inflammatory response, and compound symptoms occur such as for example, degenerative changes in cartilaginous tissue, inflammation of the synovial membrane, and other joint and joint pain attributable to inflammation due to wear and mechanical irritation of the joint cartilage, along with inflammatory responses. As the composition of the present invention contains as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt, it has multiple cartilage protection effects from mechanical irritation, inhibition of degenerative changes in cartilage caused by wear and inflammation, repair in

3 0 injuries due to cartilage trauma and suppression of joint tissue inflammation and pain. As a result, the composition of the present invention is capable of inhibiting the progress of a cartilage disease, and alleviating, ameliorating and / or curing symptoms. Furthermore, the composition for treating a cartilage disease of the present invention has the effect of improving joint function by alleviating, ameliorating and / or curing its symptoms. Improved joint function refers to increased joint range of motion, increased movement performed during 10 activities of daily living, and the like.

When applying the cartilage regeneration composition of the present invention as a fill in a cartilage defect, as it is difficult to apply the composition with a syringe if viscosity 15 is high, a pressurized or motorized syringe may be used. . The composition may also be applied to a cartilage defect, for example with a spatula or stick, without the need to use a syringe or the like. In the case of injection with a syringe, preferably a 16 to 18G needle is used, for example. In the case of applying the composition for treating a cartilage disease of the present invention by injection into a joint, preferably an 18 to 27G needle is used.

The amount of composition applied for regeneration

of cartilage of the present invention is determined according to the size of the orifice (or holes) formed in the cartilage trauma injury or cartilage defect where it is applied and, although there are no specific limitations,

30 of injecting directly into a cartilage defect, for example, the amount applied will preferably be from 0.05 to 10 mL and more preferably 0.1 to 2 mL. The application of a cartilage trauma injury preferably consists of injection to adequately fill the total void 5 of the affected area. In the case of application of the composition for treating a cartilage disease of the present invention by injection into a joint, the dose will be suitably determined according to the amount of synovial fluid in the joint into which the composition will be injected and although there is no Specific limitations, when administered to a knee joint or shoulder joint of a human, the dose will normally be from 1 to 5 mL and more preferably 2 to 3 mL. In addition, the administration method may consist, for example, of administration 15 in five consecutive administrations at one week intervals, followed by continuous administrations every 2 to 4 weeks. Although there are no specific dose limitations, the dose may be adjusted accordingly according to symptoms and effects. For example, a method of administration may be adopted in which administration is suitably continued once every two weeks or once a month. Because alginic acid is not inherently present in the body, animals do not have an enzyme capable of specifically degrading alginic acid. Although alginic acid is usually gradually decomposed by hydrolysis in an animal's body, as its decomposition in the body is slow compared to polymers such as hyaluronic acid, long-term effects can be expected when administered in

30 a joint. The composition for the cartilage regeneration or treatment of a cartilage disease of the present invention contains as an active ingredient thereof an alginic acid lower endotoxin monovalent metal salt.

The inventors of the present invention have for the first time found that alginic acid itself demonstrates regenerative and therapeutic effects on cartilaginous tissue and joint tissue when alginic acid is administered to a body joint. The fact that 10 contains alginic acid as an active ingredient means that alginic acid is present in an amount that allows it to demonstrate regenerative and therapeutic effects on cartilaginous and joint tissue when applied to an affected area, and that the amount is preferably 15. of at least 0.1% w / v or more of the entire composition, more preferably 0.5% w / v or more and particularly preferably 1 to 3% w / v.

The composition for cartilage regeneration or treatment of a cartilage disease of the present invention.

The invention may also contain commonly used components.

in pharmaceutical substances, such as other commonly used pharmaceutically active ingredients such as stabilizers, emulsifiers, osmotic pressure adjusting agents, buffers, isotonic agents, preservatives, analgesics or dyes, if necessary.

Furthermore, in one aspect of the present invention, the composition of the present invention does not contain a component that demonstrates pharmacological action on cartilage or joint tissue other than an endotoxin monovalent metal salt.

30 lower alginic acid. A composition containing as an active ingredient thereof only a monovalent lower endotoxin metal salt of alginic acid is also capable of demonstrating effects suitable for cartilage regeneration or treatment of a cartilage disease.

In addition, the composition for cartilage regeneration or treatment of a cartilage disease of the present invention may also contain a cell growth promoting factor, examples of which include BMP,

FGF, VEGF, HGF, TGF-β, IGF-1, PDGF, CDMP, CSF, EPO, IL and IF. These factors may be produced by a recombination technique, or may be purified from a protein composition.

Furthermore, in one aspect of the present invention, the

The composition of the present invention does not contain such growth factors. Even when it does not contain growth factor, however, cartilage regeneration is adequately satisfactory, and safety is greater than when cell growth is aggressively promoted.

2 0 9. Method of treatment

In addition, the present invention provides a method of treating a cartilage trauma injury and a method of treating a cartilage disease using the cartilage regeneration composition.

2 5 treatment of a cartilage disease of the present

invention as described above.

The term "treating a cartilage trauma injury" or "treating a cartilage disease" is as explained previously in section 1 entitled

3 0 "Introduction". There are no specific limitations on the method for applying the cartilage regeneration composition of the present invention to a cartilage trauma injury, and the composition can be applied, for example, by directly injecting a cartilage defect with a specific syringe, gel pipette or injector, both arthroscopically and endoscopically. Alternatively, the composition may be injected directly into a cartilage defect with a specific syringe, gel pipette or injector and the like after exposure of the affected area by a known surgical technique such as arthrotomy using a parapatellar mediai approach.

In addition, concomitant drugs, including 15 antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin or amphotericin B, or antiinflammatory drugs such as aspirin, non-steroidal anti-inflammatory drugs (NSAIDs) or acetaminophen as well can be

20 administered before, during or after application of the

composition of the present invention in a cartilage trauma injury. Such drugs may also be used by mixing in the composition of the present invention.

In addition, one or more holes may be formed in a cartilage trauma injury, and the composition of the present invention may be injected into the holes formed. In addition, the composition may also be used in the same manner by forming one or more holes in a cartilage defect.

For example, in the case of a technique involving exposure of an area affected by a surgical procedure, various defects (defects of full thickness) having a comparatively small diameter of, for example, about 1.5 mm, and extending to the subchondral bone 5 may be formed using an electric drill or steel wire and the like, in a cartilage defect where residual cartilage is present, prior to injection of the composition of the present invention, followed by injection of the composition therein. defect. As a result of the formation of full thickness defects, bone marrow bleeding occurs, which allows bone marrow cartilage precursor cells to migrate to the cartilage defect. Cartilage regeneration is promoted by the effects of migrated cartilage precursor cells and the composition of the present invention, thereby making it possible to increase the function of the entire cartilage.

Alternatively, partial defects that have a small comparative diameter of, for example, about 1.5 mm but do not extend to the subchondral bone may

2 0 be formed in a cartilage defect where cartilage

is present, followed by applying the composition of the present invention to such defects. In the case of partial defect formation, there is no bone marrow bleeding in the defect and no infiltration of cartilage precursor cells into the bone marrow. Also in this case, the effects of the composition of the present invention are demonstrated by applying the composition to small diameter holes, cartilage regeneration is satisfactory, and the function of the entire cartilage may be

3 0 improved. These techniques are effective when residual cartilage is present in the cartilage defect.

10. Kit for cartilage regeneration and treatment of cartilage disease

In addition, the present invention provides a kit for cartilage regeneration or treatment of cartilage disease. Such a kit includes the composition for cartilage regeneration or treatment of a cartilage disease of the present invention as described above, a crosslinking agent, syringe, gel pipette, specific injector, instructions, and the like. A specific preferred example of a kit is that a monovalent alginic acid metal salt is sealed in a syringe housing composed of two compartments formed integrally divided by a division, and a solution in the form of saline or a solution which It contains a calcium ion crosslinking agent such as CaCl2, is sealed in the other compartment, and is composed in such a way that the division between the compartments can be easily penetrated at the time of use to allow the contents of both. the compartments are used by mixing and dissolving at the time of use. Another example of a kit is that a solution of a monovalent alginic acid metal salt is sealed in a pre-filled syringe, which allows it to be administered directly at the time of use without the need for a preparation procedure. Another example is a kit in which an alginic acid solution and a crosslinking agent are sealed in separate syringes and packaged together in a single package. The "composition for cartilage regeneration or treatment of a cartilage disease", "crisscrossing agent" and "syringe" are as previously explained. In addition, cells may be embedded in the composition containing a monovalent metal salt of alginic acid as previously described. In addition, the kit may also contain concomitant drugs, including antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamycin, neomycin or amphotericin B, or anti-inflammatory drugs such as aspirin, non-steroidal anti-inflammatory drugs (NSAIDs). ) or acetaminophen.

Using this kit enables regenerative cartilage therapy and therapy of a cartilage disease to be performed with ease.

In addition, all publications cited in this

In the specification, for example, prior art documents, open patent applications, patent publications and other patent documents, are incorporated in their entirety in the present specification as references. Further, the present specification incorporates the contents of the specifications of Japanese Patent Application No. 2007-41520 and Japanese Patent Application No. 2007-277005, which serve as the basis for the claims of the present application.

Although an explanation is given below

By way of examples of this specification, the present invention is not limited to such examples.

Example 1 3 0 Preparation of Sodium Alginate Solution In the present example, two types of sodium alginate consisting of purified sodium alginate (Kimica Corp., Mochida International Ltd., Sea Matrix (sterilized), Serial No. 0) were used. B5Y01) and unpurified food grade sodium alginate (also called commercial grade sodium alginate, Wako Pure Chemical Industries, Ltd., sodium alginate 500, 199-09961). Purified sodium alginate was sterilized and lyophilized. The food grade sodium alginate was filter sterilized with a filter having a pore diameter of 0.22 μιη.

When endotoxin levels were measured using a commercially available LAL test kit (Limulus Color KY Test Wako, Wako, Japan), the endotoxin level of purified sodium alginate was 5.76 EU (endotoxin units) / g, and that of food grade sodium alginate was 75,950 EU / g, thus indicating that the endotoxin level of purified sodium alginate was much lower than that of food grade sodium alginate. Specifically, purified sodium alginate was subjected to endotoxin reduction treatment. In addition, the heavy metal content of the purified sodium alginate was 20 ppm or less, the lead sulphate content was 0.98% or less, and the arsenic content was 2 ppm or less.

In addition, concentrations of 1% w / v and 2% w / v of sodium alginate solutions from each sodium alginate were prepared using Milli-Q sterile filtration water. The viscosity of each alginate solution concentration of

30 Sodium at 20 ° C was then measured using a rotational viscometer (cone and plate type, TVE-2 OLT, Toki Sangyo Co., Ltd., Japan). Rotation speeds were 1 rpm when 1% sodium alginate solutions were measured and 0.5 rpm when 2% sodium alginate solutions were measured and the measuring ranges consisted of M when measured. 1% sodium alginate solutions, and 5 M when 2% sodium alginate solutions are measured. Results are shown in Table 1.

Table 1

Acid Conc. Viscosity (rtiPa.s) Ia 2 1 3a Average Deviation measured measure standard measure Grade ali¬ 1 533, 5 537, 0 531, 5 534, 0 2, 27 mentic Grade ali¬ 2 5. 377.0 5,336.0 5,325.0 5,346.0 22.38 ment Purifi¬ 1 435.4 434, 1,429.3 432, 9 2, 62 Purifi¬ 2 5,359.0 5,496.0 5,488, 0 5,447.7 62, 78 market As shown in Table 1, the viscosity of the alginate

of purified sodium was about 440 mPa.s for the 1% w / v solution and about 5,400 mPa.s for the 2% w / v solution. The viscosity of the food grade sodium alginate was about 5300 mPa.s for the 1% 15 w / v solution and about 5300 mPa.s for the 2% w / v solution. Based on the results for both groups, it was found that the viscosity of each solution of the purified sodium alginate and food grade sodium alginate used in the present example is about 400 to 600 mPa.s at a concentration of 1%. % w / v of about 5,000 to 6,000 mPa.s at a concentration of 2% w / v.

Physical properties were confirmed for food grade purified sodium alginate solutions having concentrations of 1, 2 or 3% w / v. When several drops of each concentration of sodium alginate solutions were applied from below to an inverted plastic plate, although most 1% w / v sodium alginate solutions (viscosity: approximately 400 to 600 10 mPa.s) dripped of the plaque within a few seconds due to gravity, some of the sodium alginate remained attached to the bottom of the plaque. Based on this result, if a composition containing a monovalent alginic acid metal salt had a viscosity of about 400 to 600 15 mPa.s or more, it was suggested that the effects of the present invention would be obtained, since the composition has adhesion and the property of remaining in the affected area. In contrast, sodium alginate solutions that have a concentration of 2% w / v (viscosity: approximately 5,000

20 to 6,000 mPa.s) did not slip from the plate and remained adhered to the plate for at least about one minute. Even after some of the solutions dripped from the plate, a large amount of the sodium alginate remained attached to it. Sodium Alginate Solutions That

25 having a concentration of 3% w / v remained attached to the plate longer than 2% w / v solutions.

On the other hand, regarding the ease of manipulation of the composition, the 3% w / v sodium alginate solutions needed some time to dissolve in Milli-Q water and although filling in the pipette and syringe was somewhat difficult. , the pipette and the syringe were able to be triggered. The 1% and 2% w / v sodium alginate solutions were easily manipulated.

Here, as the sodium alginate used herein was believed to be similar to the sodium alginate solution with a concentration of 1% and a viscosity of 570 mPa.s used in Experiment 10, the viscosity of the 3% w / v sodium alginate solutions. sodium was determined to be about

20,000 mPa.s. Thus, it has been suggested that the viscosity of the composition containing a monovalent metal salt of

alginic acid is preferably about 20,000 mPa.s or less with respect to ease of handling when using a pipette or syringe.

Based on the above results, when it became the viscosity of sodium alginate solutions from 5,000 to

6,000 mPa.s, easier preparation and handling was obtained, and this viscosity was indicated to be suitable for use as a composition for cartilage regeneration or for treatment of cartilage disease. At

In clinical practice, there are many cases in which the cartilage trauma injury is facing down or to the side, for example, when handling a cartilage trauma injury on the surface of a femoral joint, for example. As a result of adjusting the viscosity of the composition of the present invention, the composition has been shown to be capable of being used in a wide range of various forms of cartilage injury, even with regard to cartilage injury involving such a difficult procedure. . In addition, the concentration can be adjusted to about 2% w / v using Milli-Q water to give a viscosity of 5,000 to 6,000 mPa.s for the purified sodium alginate solution used. in the present example.

Example 2

Transplant Cell Production

Bone marrow mesenchymal stromal cells (BMSC) were isolated and cultured to obtain transplantation cells. BMSCs include erythropoietic cells and the like, as well as bone marrow 10 mesenchymal stem cells. Ten mL of bone marrow were collected from the tibia of 4-month-old Japanese white rabbits, followed by washing twice with Ca-Mg-free PBS (Gibco BRL Lab.) And suspension in DMEM-High Glucose (DMED-HG, Sigma Chemical, St. Louis, MO). Blood clots were removed with a cell strainer having a pore diameter of 70 μιη (Falcon Co., Ltd.). The cells were then incubated during humidification at 37 ° C and 5% CO 2 in a 100 mm culture dish containing a culture medium consisting of DMEM-HG, 10% fetal bovine serum (FBS, Gibco, Life Technology, 20 Grand Island, NY) and 1% antibiotics (Penicillin-Streptomycin-Fungizona Concentrates 100X, Cambrex Biosciences, Walkersville, MD). Culture medium was changed every three days, and nonadherent cells were removed. After culturing the adherent cell monolayer for 10 to 14 days, the cells were removed with trypsin-EDTA (10 mM, Sigma, GB) and counted, followed by subculturing every three days.

Example 3 Production of Alginate Blood Cells The cells obtained in Example 2 were suspended at 2.5 x 10 7 cells / mL in a sodium alginate solution adjusted to a concentration of 2% w / v with Milli-Q sterile filtered water. The suspension was gelled by dripping in CaCl2 solution with a pipette and, after twice washing the microcapsules formed 10 minutes later with PBS without Ca-Mg, the microcapsules were washed once with DMED-HG. The resulting blood cells contained 1 x 106 cells per 40 μΐ per cell.

Cells were collected from alginate globules

by washing three times with PBS and incubating at 370 ° C and 5% CO 2 in 50 mM EDTA (Gibco BRL Laboratories), followed by centrifugation for 5 minutes at 1,500 g 10 minutes later to collect the cells.

Example 4

Calcium Toxicity on Alginate Blood Cells Method

Survival rates of alginic acid encapsulated cells by CaCl2 solution drip were measured

2 0 using Cell Count Kit 8 (CCK-8, Dojindo

Laboratories, Tokyo, Japan). The cells obtained in the Example

2 were suspended at 2.5 x 107 cells / mL in a sodium alginate solution adjusted to a concentration of 2% w / v and dripped at concentrations of 50, 100, 200 and 400 mM.

CaCl2 solution according to the procedure of Example 3, and immersed for 15 minutes to obtain globules containing 1 x 10 6 cells per 40 μΐ per globule. After washing the alginate globules twice with PBS, the cells in the globules were collected using the method

30 described in Example 3, and then suspended in DMED-HG. Cells from each group were seeded in a 96-well plate and incubated for 1 hour, followed by the addition of 20 μΐ CCK-8 solution to each well and incubation for a further 4 hours. Cell survival rates were obtained by measuring absorbance at 450 nm using a microplate reader (Bio-Rad Japan Life Science Research, Tokyo, Japan).

Results

Cell survival rates at each CaCl2 solution concentration are shown in FIG. 1.

Cell Survival Rates In Alginate Blood Cells

decreased in a calcium-dependent manner, and decreased significantly starting at a concentration of 200 mM. Thus, it was demonstrated that CaCl2 has cytotoxicity. In addition, it was found that

It was appropriate to make the concentration of calcium chloride contacting sodium alginate about 100 mM to minimize effects on cells and allow sodium alginate to gel as quickly and firmly as possible.

2 0 Example 5

Comparison of cell survival rates in blood cells of

alginate

Method

The survival rates of cells in blood cells from

alginate were compared for purified sodium alginate submitted to endotoxin reduction treatment and for food grade sodium alginate not subjected to endotoxin reduction treatment. Each of the alginate globules was produced by suspending the obtained cells.

30 in Example 2 in 2% w / v sodium alginate solution according to the procedure of Example 3, followed by dripping into 100 mM CaCl2 solution. Each globule was made to contain 1 x 106 cells per 40 μΐ per globule. Both types of alginic acid capsules were cultured 5 for 0, 1, 2, 3, 7, or 14 days in DMED-HG containing 10% FBS and 1% antibiotic. Cells were collected from each capsule according to the method described in Example 3, and viable cell numbers were counted using the CCK-8 kit.

Results

Results are shown in FIG. 2. The number of

remaining viable cells was significantly larger when using purified sodium alginate solution subjected to endotoxin reduction treatment as compared to the use of food grade sodium alginate solution not subjected to endotoxin reduction treatment in Io, 3rd and 7th days. It has been confirmed that sodium alginate solution undergoing endotoxin reduction treatment offers the advantage, for example, of having less toxicity to cells, particularly at the beginning of the procedure (within 7 days), as compared to solutions that do not undergo endotoxin reduction treatment.

Example 6

In vitro alginate globule cultivation

Method

(Cultivation)

Blood cells containing 1 x 106 cells per, 40 μΐ per cell, similar to those in Example 5, were produced according to Example 3 for sodium alginate.

30 and for food grade sodium alginate, respectively. Individual blood cells were placed in each well of 24-well culture plates and grown in

1 mL of the standard culture medium described below. Specifically, the standard culture medium used consisted of DMEM-HG containing 100 pg / mL sodium pyruvate (ICN Biochemicals, Aurora, OH), 40 pg / mL proline (ICN Biochemicals, Aurora, OH), 50 pg. / ml ascorbic acid 2-phosphate (Wako, Osaka, Japan), 1 x 10 7 M dexamethasone (ICN Biochemicals, Aurora, OH), ITS Plus Mix 1% (Sigma-Aldrich, St. Louis, MO) 1% antibiotics and 10 ng / mL recombinant human growth transforming factor β3 (R&D System, Minneapolis, MN) dissolved in 4 mM HCl containing 1 mg / mL bovine serum albumin. The culture plates were incubated at 37 ° C and the medium changed every three days.

Real-time RT-PCR RNA Analysis

After cultivation for 14 days, total RNA was removed from homogenized cells and collagen type I, II and X gene expression, Agrecana and Sox 9 were analyzed. All experiments were performed by conventional methods.

Specifically, RNA yield was determined by measuring absorbances at 260 and 280 nm. Next, cDNA was synthesized from 0.05 pg RNA using the ImProm-II ™ Reverse Transcription System (Promega, Madison, WI) 25 according to the manual. At this time, the total RNA binding product and a random primer were denatured for 5 minutes at 70 ° C, immediately followed by cooling for 5 minutes in ice water and reverse transcribed for 60 minutes at 42 ° C using

30 ImProm-II ™ reverse transcriptase. The resulting cDNA was then diluted with PCR grade water (Roche Diagnostics, Indianapolis, IN) to adjust the concentration to below 40 ng / μΐ. PCR was then performed in a reaction volume of 20 μΐ and monitored using the DNA Engine Opticon ™ 2 continuous fluorescence detection system (Bio-Rad Laboratories, Hercules, CA). Signals were detected with the "SYBR Green qPCR" Kit (Finzyme, Espoo, Finland) using DNASIS-designated gene-specific primers (Hitachi Software Engineering, Tokyo, Japan).

More specifically, the primers used are the

indicated below.

Rabbit Collagen Type I: (5'-3 ') TAAGAGCTCCAAGGCCAAGA (SEQ ID NO: 1) and (3'-5') TGTACCTACTCCTTTGACCG (SEQ ID NO: 2).

Rabbit Collagen Type II: (5'-3 ')

AGAGACCTGAACTGGGCAGA (SEQ ID NO: 3) and (3'-5 ') ACCACGATATGAGGCACAGTTT (SEQ ID NO: 4).

Rabbit Collagen Type X: (5'-3 ') GCCAGGACCTCCAGGACTAT (SEQ ID NO: 5) and (3'-5')

20 CTTTGGACCTGTTGTCCCT (SEQ ID NO: 6).

Rabbit Agecana: (5'-3 ') GAGGTCGTGGTGAAAGGTGT (SEQ ID NO: 7) and (3'-5') TGACAGT C C ATGGGGT AGGT (SEQ ID NO: 8).

Rabbit Sox 9: (5'-3 ') AAGGGCTACGACTGGACGCT (ID.

SEQ No. 9) and (3'-51) GTGCAGTTCGCCGGGT (SEQ ID NO: 10).

After an initial denaturation step for 10 minutes at 95 ° C, cDNA products were amplified by 40 cycles of PCR. Each cycle consisted of a denaturation step for 10 seconds at 94 ° C, a

30 girdling for 20 seconds at 58 ° C and an stretching step for 30 seconds at 72 ° C. Data were analyzed using Opticon Monitor ™ software (Bio-Rad Laboratories, Hercules, CA). The value obtained for each sample when the fluorescence intensity reached 5 0.03 was determined to be the Ct (cycle threshold) value. This value was selected by confirming that all curves were in exponential amplification phase within this range. The relative expression level of each gene was calculated using the Ct 10 comparative method modified from the Ct values of each target gene and the reference gene (GAPDH).

Coloring

Blood cells were washed with PBS after 21 and 28 days of culture, and after 24 hour fixation with 10% phosphate buffered paraformaldehyde 15, the blood cells were embedded in paraffin. The globules were cut in 5 μιη sections from the center of the globules, followed by H-E staining and safranin-0 staining according to standard methods. In addition, type I, II 20 and X collagen formation was confirmed with anti-type I, anti-type II (Fuji Pharm. Lab., Toyama, Japan) and anti-type X (Sigma, St. Louis, MO).

Results

Results of real-time RT-PCR RNA analysis are shown in FIG. 3. In addition, staining results are shown in FIG. 4. FIG. 4A and FIG. 4B show the results when using purified sodium alginate, while FIG. 4C and FIG. 4D show results when using food grade 30 sodium alginate. In addition, FIG. 4A and FIG. 4C show results for 21 days of cultivation, while FIG. 4B and FIG. 4D show results for 28 days of cultivation. In addition, FIG. 4A to FIG. 4D show, respectively, from left to right, the results for 5 H-E staining, safranin-0 staining, and anti-type I, anti-type II, and anti-type X anti-collagen antibodies.

Referring to RT-PCR results (FIG. 3), increases in collagen type II, agrecana and Sox 9 were observed, which indicate cell differentiation into 10 cartilage when using purified sodium alginate or grade sodium alginate. food. A comparison of both types revealed that cultivation with purified sodium alginate generated significantly higher levels of agrecana and Sox 9.

In addition, with reference to the staining results

(FIG. 4), an extracellular matrix was produced which was stained with safranin-0 and collagen-type immunostaining.

II, which indicates cartilage differentiation for both types of alginate globules, and cartilage differentiation was observed.

Example 7

Rabbit Cartilage Repair Template

Procedure

Forty female Japanese white rabbits (body weights: 2.6 to 2.9 kg) were anesthetized with isoflurane in O2 gas and intravenous pentobarbital injection (0.05 mg / kg), followed by intramuscular injection of antibiotic (Penicillin G , Meiji-Seika, Japan) and leg trichotomy. An anteromedial incision of 2 cm was made in the skin and the

3 The trochlear groove was accessed using a parapatellar mediai approach. Osteochondral defects (diameter: 5 mm, depth: 2 mm) were created in the femoral trochlea using an electric drill (Rexon, Japan). The knees were then irrigated with saline, the absence of 5 bleeding in the defects was confirmed, and the defects were allowed to dry.

In the present example, the experiment was performed by dividing the animals into five groups.

A) Control Group (empty)

B) Food grade alginate group (without cells)

C) Food Grade Alginate Group + Cells (2.5 x 107 / ml)

D) Purified alginate group (without cells)

E) Purified alginate group + cells (2.5 x 10 7 / ml)

Defects remained untreated in the control group (A). In addition, a 2% w / v food grade sodium alginate solution was applied to the defects in the food grade alginate group (B) (without cells). The 2% w / v solution of purified sodium alginate was applied to the defects in the purified alginate group (D) (without cells). In addition, the cells obtained in Example 2 were suspended in either 2% w / v food grade sodium alginate solution or 2% w / v solution of purified sodium alginate, and applied to articular cartilage defects in the alginate group. food grade + cells (C) and in the purified alginate group + cells (E), respectively. Rabbit autologous cells prepared according to the method described in Example 2

30 were used for the cells at this time. The reason for making the concentration of sodium alginate solutions 2% w / v is that the viscosity can be adjusted to a level of 5,000 to 6,000 mPa.s, appropriate to the procedure based on the results of Example 1.

Rabbits were immobilized with defects turned

above, and the composition of the present invention was applied to the defects using a gel pipette.

As the viscosity of the sodium alginate solution was adequate in groups (B) to (E), sodium alginate solutions did not flow out of the defects despite conditions that facilitate flow due to synovial fluid. Subsequently, approximately 0.5 mL of a 100 mM CaCl2 solution was slowly and continuously applied over 10 seconds to graft surface 15 using a 27G syringe. The graft surface layer immediately gelled, and the cells did not leave the affected area. The CaCl 2 solution was washed with saline. No additional immobilization was required, and the affected area was sutured after the procedure. The rabbits

20 were able to move freely.

The rabbits in question were sacrificed by intravenous injection of an excessive dose of pentobarbital at 4 weeks or 12 weeks after the procedure. The distal ends of the femurs were removed with a chainsaw. FIG. 5 shows photographs taken at the time of the procedure.

Global Observations

The overall appearance was observed macroscopically and punctuated. Overall appearance was scored according to the criteria of FIG. 6 with reference to the method of Gabriele, G. et al. (Biomaterial, 21 (2000), 2,561-2,574).

Coloring

Subsequently, the samples were fixed with paraformaldehyde, decalcified and embedded in paraffin.

Sections located 5 μιη from the center of the defect were stained with safranin-O, H-E staining and immunostained with anti-collagen type I and anti-collagen type II. The scoring system described in FIG. 7 was used to evaluate newly formed cartilaginous tissue, and tissue 10 was evaluated microscopically. Independent blind observers scored.

Mechanical strength measurement

The mechanical strength of the affected area was measured using an indentation test. The samples were clamped firmly with the patellofemoral joint facing up, and the test was performed at room temperature. The indentator was automatically moved toward the center of the regenerated cartilage and the displacement (mm) was recorded relative to the load (N). The thickness of the regenerated tissue was measured from the histological sections. Young's modulus of the linear region of the load-displacement curves was then obtained.

Results

Staining results are shown in FIG. 8 to FIG. 11

As a result of H-E staining, safranin-0 staining, and anti-collagen type II immunostaining, the most prominent formation of hyaline cartilage and type II collagen compared with the other groups was

30 confirmed in the purified alginate + cells (E) group (FIG. 11) at an early stage 4 weeks after the procedure. It was observed that approximately 80% of cartilage was regenerated within 12 weeks after the procedure. Subchondral bone formation was extremely favorable based on H-E staining results. Safranin-0 staining revealed proteoglycan formation, and formation of an extracellular matrix can also be confirmed. On the other hand, little or no fibrous cartilage formation based on H-E staining and anti-collagen type I immunostaining was observed.

The purified cell-free alginate group (D) (FIG. 10) demonstrated favorable formation of hyaline cartilage, type II collagen, and subchondral bone when compared to the food-grade alginate + cell group (C) (FIG. 9). In group (D), in which no cells were embedded, it was surprisingly found that cartilage regeneration was achieved by hyaline chondrocytes. In addition, it was also unexpectedly found that group (D),

20 in which no cells were embedded, demonstrated a

superior ability to regenerate cartilage trauma when compared to group (C) in which cells were embedded.

On the other hand, very little type II cartilage and collagen neogenesis was observed in the control group (A) (FIG. 8), in which the defects were left untreated.

The evaluation results obtained by macroscopically scoring the overall appearance (Macro) and the evaluation results obtained by

The observations based on the staining described above (Histological) are shown in FIG. 12

The total scores obtained by combining the Macro and Histological scores at week 12 were 22.71 for the purified alginate + cells (E) group, 19.57 5 for the purified (without cells) alginate group (D), 14.75 for food-grade alginate group + cells (C), 10.25 for food-grade alginate group (without cells) (B), and 8.43 for control group (A) (empty) ). Thus, the purified alginate group + cells (E) showed the highest score, followed by the purified alginate group (without cells) (D) and the food grade alginate group + cells (C), in that order. It was completely unexpected that group (D), in which no cells were embedded, generated a higher total score and thus demonstrating superior ability to regenerate cartilage in cartilage lesions when compared to group (C), in which cells were soaked.

Scoring results for both assessment

The macroscopic overall appearance (Total macro) and stain evaluation (Total Histological) were the highest in the purified alginate + cells (E) group, as described above, and the next highest score was observed in the group. of purified alginate (without 25 cells) (D).

Observing the Macro evaluation parameters, groups (D) and (E), in which purified alginate was used, were superior for all margin integration parameters (new tissue in relation to the native cartilage), surface uniformity of the cartilage, cartilage surface, degree of cartilage filling and color, opacity or translucency of neocartilage, when compared with groups (B) and (C), in which food grade alginate was used.

Observing the histological evaluation parameters,

groups (D) and (E), in which purified alginate was used, showed higher scores than groups (B) and (C), in which food-grade alginate was used, for the predominant tissue nature parameters 10. , surface regularity, structural integrity and homogeneity, thickness, union to adjacent cartilage, degenerative changes in adjacent cartilage, and inflammatory response.

Based on these findings, the composition of the present invention, as represented by groups (D) and (E), demonstrated extremely favorable formation of chondrocytes and cartilage tissue in a cartilage trauma, including the formation of hyaline cartilage, type II collagen. and subchondral bone. Little cartilage formation was observed

20 fibrous.

The union of regenerated tissue to host tissue was also favorable, there was little degeneration or inflammation in the adjacent cartilage, and bioaffinity was determined to be high.

Thus, it was confirmed that the composition of the

The present invention effectively promotes cartilage regeneration in a cartilage trauma injury.

The results of the mechanical strength measurement for the purified alginate groups (D) and (E) are shown in FIG. 13. «

As a result of measuring the mechanical resistance for the purified alginate groups, the mechanical resistance in the purified alginate + cells group (E) was a Young modulus of 8 versus a Young modulus of 10 in normal 5 cartilaginous tissue, indicating that Thus, the resistance was recovered almost to normal, from a state without injury. This finding also supported the claim that the cell-soaked composition of the present invention has superior mechanical strength, and is favorable with

relation to strong hyaline cartilage regeneration and subchondral bone formation.

Example 8

Male corpse model submitted to a

proper treatment

Method

A male human cadaver who had undergone adequate treatment was fixed with formalin. There was no knee instability or deformity at room temperature. The lateral condyle of the femur was

20 exposed using a parapatellar mediai approach. THE

Articular cartilage was smooth and no degeneration or deterioration was observed. A defect of total cartilage thickness measuring 10 mm x 20 mm wide and 5 mm deep was produced in the section with the maximum height of the

condyle mediated using various types of punctures and then sutured. An arthroscope was inserted at an angle of 30 ° from the anterolateral side. All surgical instruments were inserted from the anterolateral side. After saline has been applied to the affected area, the remaining fluid in the

3 The joint was drained, followed by wiping with a dry cotton swab. A 2% w / v solution of trypan blue stained (cellless) purified sodium alginate with a viscosity of 5,000 to 6,000 mPa.s was slowly injected into the cartilage defect with a 518G needle syringe. Although the affected area was facing downwards at the time of the procedure, the composition of the present invention did not leave the lesion and remained there. Ten mL of 100 mM CaCl2 solution was applied to the affected area to gel the surface. The knee joint was adequately washed by refluxing with saline, and the affected area was filled with 20 mL saline to prevent dryness.

After the procedure, the knee was manually extended and flexed to a range of 0 to 120 °, 200 times each, at six-hour intervals. The affected area was evaluated 24 hours after the procedure.

Results

Photographs revealing this experiment are shown in FIG. 14. FIG. 14A is a photograph showing the creation of the cartilage defect. In addition, FIG. 14B shows a stained solution of sodium alginate transplanted into the cartilage defect. FIG. 14C shows the surface of sodium alginate being gelled (cured) by application of CaCl2 solution. Finally, FIG. 14D 25 shows the results of observing joint movement 24 hours after surgery.

This experiment was performed to confirm whether the composition of the present invention can be transplanted if a large defect has been created not only in

3 0 rabbits, but also in a human corpse. As shown in the photograph of FIG. 14B, the sodium alginate solution did not leak out of the affected area even if the surface of the sodium alginate solution was not gelled after injection into the defect. In addition, the sodium alginate solution remained in the defect even after the joint was moved after surgery and observed 24 hours later after gelling the surface of the sodium alginate solution. It was surprising to find that a composition of this form was able to remain in the defect at the site subjected to the harsh conditions of applying a violent charge and movement. Based on this finding, it has been found that the composition for cartilage regeneration or treatment of a cartilage disease of the present invention has physical properties that allow it to be applied to a wide range of clinical applications with respect to the various forms of cartilage injury. cartilage and conditions of use.

Example 9

Technique for forming one to several holes in an injury to

cartilage

(1) First example

In cases where cartilage remains in a cartilage trauma injury or cartilage defect, one to several comparatively small diameter full thickness defects having a diameter of about 1.5 mm and a depth of about 5 mm. 10 mm and extending to the subchondral bone are produced in a cartilage trauma or residual cartilage injury using an electric drill according to the method of Examples 7 and 8. After slowly injecting into these holes a solution of purified sodium alginate (without cells) with a viscosity of 3,000 to 4,000 mPa.s with an 18G needle, 1.0 mL of 100 mM CaCl 2 solution is applied to the surface of the sodium alginate solution injected into the defect to gel the surface. As a result of producing the full thickness defect (or defects), bleeding occurs from the patient's bone marrow, which allows bone marrow cartilage precursor cells to migrate to the cartilage defect. Cartilage regeneration is promoted by the effects of migrated cartilage precursor cells and the composition of the present invention, thereby making it possible to improve the function of the entire cartilage.

(2) Second example

A partial defect that does not extend to the subchondral bone is produced in a cartilage trauma injury or a cartilage defect in which residual cartilage is present, similar to the first example described above. A purified (cellless) sodium alginate solution with a viscosity of 2,000 to 3,000 mPa.s and a 100 mM CaCl 2 solution are then applied in the same manner as the first example. As there is no bleeding in the defect by the patient's bone marrow, there is no infiltration of cartilage precursor cells in the individual's bone marrow. However, also in this case, as a result of the application of the composition to the small diameter holes, the effects of the composition of the present invention are demonstrated, cartilage regeneration is favorable, and the function of the entire cartilage can be improved. These techniques are effective in cases where a cartilage trauma injury covers a wide area and in those where damaged residual cartilage is present.

Example 10

Sodium Alginate Solution Adhesion Test

The relationship between viscosity and adhesion of

The composition of the present invention was examined using an aqueous sodium alginate solution (Kimica Corp.).

Method

Aqueous sodium alginate solutions 10 (Table 2) were prepared using three types of sodium alginate solutions for which the viscosity of a 1% aqueous sodium alginate solution shows a value of 110, 360 or 570 mPa.s at because they have different molecular weights. A predetermined amount of each solution was poured into a centrifuge microtube (internal diameter: 9 mm, height:

39 mm), preventing the entry of air bubbles, followed by immediate measurement of the amount of time until each solution begins to drip out of the microtube when tilted at an angle of 135 °.

2 0 At this point, the viscosities of the solutions of

Sodium alginate were measured using a type B viscometer (Toki Sangyo Co., Ltd., Japan) at a temperature of 20 ° C.

Table 2

Sample

Aqueous Concentration Solution (%) sodium alginate Viscosity 110 mPa.s 0.5 1.0 1.5 2, 0 2.5 3.0 3 60 mPa.s 0.5 1.0 1.5 2, 0 2.5 3.0 570 mPa.s 0.5 1.0 1.5 2, 0 2.5 3.0 Results

The relationships between the concentration of each sodium alginate solution and the adhesion time are shown in FIG. 15. The adhesion times of each of the three types of sodium alginate solutions increased as the concentration of the solutions became higher, and it was determined that the adhesion increased. Furthermore, a comparison of the three types of sodium alginate solutions revealed that the selection of a sodium alginate 10 solution that had a high viscosity at a concentration of 1% yielded high adhesion and achieved an adhesion time. longer.

The relationships between the viscosity of each type of sodium alginate solution and the adhesion time are shown in FIG. 16. The adhesion time became longer and greater adhesion was demonstrated as the viscosities of sodium alginate solutions increased. Thus, it was demonstrated that a constant correlation between viscosity and adhesion of a composition 20 containing a monovalent alginic acid metal salt was obtained.

Based on these findings, when applying the composition of the present invention to a slanted or downward-facing cartilage defect, and when moisture, blood and the like in the affected area were removed and the conditions of that experiment were met, the viscosity of the composition of The present invention may be adjusted based on this result. For example, the viscosity of the composition of the present invention may be adjusted to about 2,000 mPa.s or more to obtain an adhesion time of about

30 seconds, to a viscosity of about 3,000 to 4,000 mPa.s or more, to achieve an adhesion time of about 10 seconds, up to about 7,000 to 8,000 mPa.s or more to obtain a adhesion of about 20 seconds and up to a viscosity of about 8,000 to 9,000 mPa.s or more to achieve an adhesion time of about 30 seconds.

However, when it actually applies to an affected area, the adhesion time varies depending on factors such as the amount of composition injected and the shape of the injected site. Particularly, when only a small amount of the composition is injected, as factors such as surface tension also have an effect, in addition to viscosity, long-term adhesion is possible even at low viscosity.

Target adhesion time can be obtained according to the type of procedure used by adequately taking into account other factors such as the characteristics of the viscometer used for the measurement, ambient temperature, amount of embedded cells, and condition of the composition. present invention.

Example 11

Measurement of the alginate molecular weight distribution of

purified sodium

(1) Method

The molecular weight distribution of purified sodium alginate was measured by gel filtration chromatography under the conditions indicated below.

Column: TSkgel GMPWxl, 2 columns + TSkgel G2500PWxl, 1 column (Tosoh Corp.) (diameter 7.8 mm x 300 mm x 3 columns)

Column Temperature: 4O0C

Eluate: 200 mM aqueous solution of sodium nitrate Sample concentration: 0.05%

Flow rate: 1.0 ml / min Injection Volume: 200 μΐ Detector: RI (Differential Refractometer)

Standards: Pululan, glucose (molecular weights:

1,600,000, 788,000, 404,000, 212,000, 112,000, 47,300, 22,800, 11,800, 5,900, 180)

(2) Results

Table 3

Weight Sample Weight Ratio (Reference) Molecular Measurement of Average Viscosity (Mn) weight variance of an average (Mw) (Mw / Mn) aqueous solution 1% Alginate 430. 000 1,700,000 4.0 400 to 500 purified sodium mPa. (Kimica Corp., Mochida International Ltd., Sea Matrix ™ (sterile), Serial No. B5Y01) 66. 000 440,000 6, 20 20 to 100 purified sodium mPa. s (Pronova ™ SLG20, FMC Biopolimer Inc. 10

(3) Discussion The weight average molecular weight of the purified sodium alginate used in the rabbit cartilage repair model of Example 7 was 1,700,000, measured using the method described above. As indicated in Example 7, sodium alginate demonstrated regenerative effects on hyaline cartilage in the rabbit cartilage repair model, with and without cells. On the other hand, although a similar experiment was performed using lower endotoxin alginic acid (Pronova ™ LVG, currently Pronova ™ UP LVG, FMC Biopolimer Inc.), as described in Reference 5, it is revealed that fibrous cartilage forms in the case of apply only alginic acid that contains no cells in a cartilage defect. In addition, the sterile version of Pronova ™ LVG is designated as Pronova ™ SLG20, whose average weight molecular weight, determined by the method described above, was 440,000. Although Sea Matrix ™ and Pronova ™ have a common characteristic of being lower endotoxin alginic acids, their alginic acids differ in molecular weight, and it is believed that this difference leads to differences in regenerative effects on cartilage. Although viscosity can be adjusted by alginic acid concentration, in an experiment in which different concentrations of alginic acid gels (0.5 to 4%) were soaked with chondrocytes, transplanted below mouse skin and confirmed for cartilage generation. , it has been reported that alginic acid concentration has no effect on the effects of cartilage generation (Keith T. Paige et al., "Again Cartilage Generation Using Calcium Alginate-Chondrocyte Constructs", Plastic and Reconstructive Surgery, Vol. 97: 1996, pages 168-178). Thus, it is believed that the difference in regenerative effects on cartilage between Sea Matrix ™ and Pronova ™ is attributable to molecular weight. Specifically, while the use of lower endotoxin alginic acid allows for a higher bioaffinity composition with low levels of degeneration and inflammatory responses in the surrounding cartilage, also when using high molecular weight alginic acid, it has been found that it may be A cartilage regeneration composition or a therapeutic composition having extremely superior cartilage regenerative effects is obtained, allowing cartilage regeneration even without cells embedded in it. Lower endotoxin alginic acid with a weight average molecular weight of at least 500,000 or more and preferably 650,000 or more is useful for cartilage regeneration, more preferably having a weight average molecular weight of 1,000,000 to 2,000. It is particularly preferred that it has a weight average molecular weight of from about 1,500,000 to 2,000,000.

Example 12

Rabbit Osteoarthritis Model (Anterior Cruciate Ligament Resection (ACL) Model)

(1) Method

An OA model was created in both joints of the

knee of female Japanese white rabbits (body weights: 2.6 to 2.9 kg) according to the method of Vignon, E. et al. (Vignon, E., Bejui, J., Mathieu, P., Hartmann, JD, Ville, G., Evreux, JC, et al., "Histological cartilage changes in a rabbit model of osteoarthritis", J. Rheumatol. 1987: 14 (Special No.): 104-6). Three animals (6 knees) were distributed in the following four groups:

A) Control group (saline)

B) Dose group of 1% sodium hyaluronate solution (molecular weight: approximately 900,000, viscosity:

approximately 2,300 mPa.s)

C) Purified sodium alginate 1% solution dose group (molecular weight: approximately 1,700,000, viscosity: approximately 500 mPa.s)

D) Sodium alginate 2% solution dose group

purified (molecular weight: approximately 1,700,000, viscosity: approximately 5,000 mPa.s)

Solutions from (B) to (D) were prepared using saline. Purified sodium alginate from (C) and (D)

was the same as the purified sodium alginate used in Examples 1 and 7 (Kimica Corp., Mochida International Ltd., Sea Matrix (sterile), Serial No. B5Y01).

After anterior cruciate ligament resection, each of the above solutions (A) through (D) was administered into the

20 Articular cavity at 4th, 5th, 6th, 7th and 8th weeks (total

5 administrations given once a week). The solutions were administered using a 27G needle penetrating the patellar tendon and injecting 0.3 ml / knee per administration. Rabbits were sacrificed at week 9

for taking tissue samples from the knee joint. Inflammation due to infections, foreign body reactions, and the like were not observed in either knee.

(2) Results

3 0 General remarks The appearance of the entire knee joint (femoral and tibial knee joint cartilage) was macroscopically observed. These results are shown in FIG. 17. In group A (saline dose group),

Several findings of osteoarthritis, including cartilage defects and osteophytes, were observed.

macroscopically. The degree of cartilage trauma (size, depth) was milder in the other groups than in group A. Macroscopic findings yielded similar results.

Coloring

Knee joint tissue samples were fixed with paraformaldehyde, decalcified and paraffin embedded. Samples were histologically evaluated by safranin-0 staining. These results are shown in FIG. 18. The upper portions of each figure indicate femoral cartilage, while the lower portions indicate tibial cartilage, and cartilage changes.

Degenerative lesions were evaluated on cartilage at both 20 locations. Decreased staining of the matrix cartilage and increased roughness of the cartilage surface were observed in group A (saline dose group). In group B (1% sodium hyaluronate solution dose group), although the cartilage surface was smoother than in group A, a decrease in color was observed. In group C (1% purified sodium alginate solution dose group) and group D (2% purified sodium alginate solution dose group), the cartilage surface was smooth and the

30 staining was lighter compared to groups A and B. In addition, residual alginic acid was present on the surface of the cartilage.

Based on the above findings, intra-articular injection of sodium alginate demonstrated action that inhibited cartilage degeneration and protected cartilage in an ACL resection OA model, and effects that were equal to or better than ACL were observed. administration of the 1% sodium hyaluronate solution used as a therapeutic drug for osteoarthritis. In addition, since sodium alginate was adhered to the cartilage surface, it was confirmed that sodium alginate demonstrates affinity with articular cartilage as well as covers and protects cartilage surfaces.

Example 13

Evaluation of the therapeutic effects of alginic acid of different molecular weights in a rabbit osteoarthritis model (cruciate ligament resection model)

previous (ACL))

(1) Method

2 0 An OA model was created in both joints of the

knee of female Japanese white rabbits (body weights: 2.6 to 2.9 kg) according to the method of Vignon, E. et al. (Vignon, E., Bejui, J., Mathieu, P., Hartmann, JD, Ville, G., Evreux, JC, et al., "Histological cartilage 25 changes in a rabbit model of osteoarthritis, J. Rheumatol., 1987: 14 (Special No.): 104-6) Five animals each (10 knees) were assigned to the following five groups:

A) Control group (saline)

B) Sodium Hyaluronate Solution 1% Dose Group (ARTZ (Trademark), Kaken Pharmaceutical Co., Ltd., Molecular Weight: Approximately 900,000, Viscosity: Approximately 2,300 mPa.s)

C) 2% Purified Sodium Alginate Solution Dose Group (Pronova ™ SLM2O, FMC Biopolimer Inc., Weight

molecular: approximately 40000)

D) Purified sodium alginate 2% solution dose group (Kimica Corp., sterile, molecular weight: approximately 1,000,000)

E) Purified Sodium Alginate 2% Solution Dose Group (Sea Matrix (Sterile), Kimica Corp., Weight

molecular: approximately 1,700,000)

Solutions from (C) to (E) were prepared using saline.

After resection of the anterior cruciate ligament, each of the above solutions (A) through (E) was administered into the joint cavity at the 4th, 5th, 6th, 7th, and 8th weeks (total of 5 administrations given once a week). The solutions were administered using a 27G needle penetrating the patellar tendon and injecting 0.3 ml / knee per administration. Rabbits were sacrificed at week 9 for tissue sampling from the knee joint. Inflammation due to infections, foreign body reactions and the like were not observed in either knee.

(2) Results

General observations

The appearance of the entire knee joint (femoral and tibial knee joint cartilage) was observed macroscopically. In order to assess the degree of

In the cartilage surface lesion, the samples were stained in ink according to the method of Choji Shimizu et al. and then scored (J. Rheumatol., Vol. 25, pages 1.813-1.819, 1998). Macroscopic findings are shown in FIG. 19. When using 5-ink stain, the boundaries between cartilage trauma lesions and normal cartilage are stained. In group A (saline dose group), several findings of osteoarthritis, including deep and wide cartilage defects and osteophytes, were observed.

macroscopically. The degree of cartilage trauma (size, depth) was lighter in the other groups than in group A. Results of macroscopic findings are shown in FIG. 20. Knee joints were observed in four locations: condyle

femoral mediai (MFC), tibial mediai plateau (MTP), lateral femoral condyle (CFL) and lateral tibial plateau (LTP). The degree of cartilage trauma was milder in groups B through E than in group A at all these sites. In addition, the degree of cartilage trauma tended to be milder in groups D and

2 0 E than in groups B and C. It is believed that the differences

inhibitory effects of degenerative cartilage alteration, protective effects of cartilage and cartilage repair effects are present as a function of differences in molecular weight of alginic acid.

Proteoglycan Staining

Knee joint tissue samples were fixed in paraformaldehyde, decalcified and embedded in paraffin. Samples were histologically evaluated by safranin-O staining. These results are shown.

30 in FIG. 21. The upper portions of each figure indicate femoral cartilage, while the lower portions indicate tibial cartilage, and cartilage changes.

Degenerative lesions were evaluated on cartilage at both locations. Decreased staining of the matrix cartilage and increased roughness of the cartilage surface were observed in group A (saline dose group). In group B (1% sodium hyaluronate solution dose group), although the cartilage surface was smoother than in group A, a decrease in staining was observed. In the sodium alginate solution dose groups (CaE groups), the cartilage surface was smooth and the color decreases were milder compared to groups A and B. In addition, residual alginic acid was present on the cartilage surface.

Global histopathological evaluation

Macroscopic observations and staining observations were comprehensively assessed by scoring according to the Toshiyuki Kikuchi et al. to evaluate the effects of administered drugs (Osteoarthritis and 20 Cartilage, Vol. 4, pages 99-110, 1996). The medial femoral condyles were evaluated at one of four levels for the 8 parameters indicated below, and the total score was used as an osteoarthritis injury score.

(I) loss of cartilage surface, (2) erosion of

cartilage, (3) fibrosis and cracking, (4) loss of colorable proteoglycan, (5) disorders in chondrocyte arrangement, (6) chondrocyte loss, (7) subchondral bone loss, and (8) formation of chondrocyte clusters .

30 AN0VA was used to test for the presence of a significant difference between groups, and subsequent comparisons between each group were made at a significance level of p <0.05 using a post hoc test.

Results are shown in FIG. 22. Osteoarthritis lesion scores 5 were significantly lower in the BaE versus group A groups. In addition, although higher effects were observed in the high molecular weight alginic acid dose groups (DeE groups) compared with the hyaluronic acid (group 10 B), the effects of the low molecular weight alginic acid dose group (group C) were approximately equal to those of the hyaluronic acid dose group.

Based on the above findings, intraarticular injection of sodium alginate demonstrated action that inhibited degenerative cartilage changes and protected cartilage in an ACL resection OA model, and effects that were equal to or better than administration were observed. of the 1% sodium hyaluronate solution used as a therapeutic drug for osteoarthritis. In particular, high molecular weight alginic acid has shown superior therapeutic effects than hyaluronic acid. In addition, although the three types of alginic acid differed in viscosity, as it was observed that alginic acid that has lower viscosity than hyaluronic acid showed equal or greater effects than hyaluronic acid, it is believed that differences in therapeutic effects are attributable to differences in substance used and molecular weight rather than viscosity differences.

In the ACL resection OA model used in this experiment, drugs were administered starting 4 weeks after ACL resection. Thus, it is believed that the decreases in osteoarthritis injury scores observed in the drug dose groups are the combined result of effects that inhibit the progression of lesions due to inhibition of degenerative cartilage changes and cartilage protection and the action of cartilage repair in cartilage lesions that have already occurred. According to the above mentioned Toshiyuki Kikuchi work cited as a reference in this experiment, OA scores are reported to reach 20 to 25 in the saline dose groups. As drug administration was started on the 4th week after ACL resection in this experiment, there is a possibility that OA scores may have decreased as a result of improved cartilage status due to drug effects following initiation of administration from of a state in which OA scores were about 20 to 25. In addition, as the score for normal joints is 8 in the rating system used in this experiment, the average OA score can be considered (11,3) in group E (alginic acid with a molecular weight of 1,700,000) approaches the score for normal joints and can be considered an extremely good score.

Example 14

Study of the acid molecular weight measurement method

alginic

It is known that different values can be obtained when measuring the molecular weight of high molecular weight substances derived from a natural origin, depending on the measurement method. According to ASTM F2064-00 (ASTM International Publication (2006); the American Society for Testing and Materials is an organization involved in international standardization and the establishment of specifications for industrial material standards and test method standards), the SEC-MALLS (Size Exclusion Chromatography with Multi-Angle Laser Light Scattering Detection) is recommended for molecular weight measurement. Therefore, a comparison was made between the molecular weight measurements of sodium alginate used in Example 13 by SEC-MALLS and by gel filtration chromatography as described in Example 11. In addition, SEC-MALLS combines the use of a detector. Multi-angle Laser Light Scattering (MALLS) with gel filtration chromatography.

(1) Method

Measurement by gel filtration chromatography was performed as in Example 11. Measurement by SEC-MALLS was performed under the conditions indicated below. Multilateral laser light scatter detector

angles: DAWN HELEOS, Wyatt Technology

Column: Shodex SB-8 06M, 2 columns (Showa Denko K.K.) Eluate: 200 mM aqueous solution of sodium nitrate Flow rate: 1.0 ml / min (2) Results

Table 4

AL17 0 AL100 AL4 0 Weight molecular weight 1,700,000 1,000. 000 410,000 average determined by gel filtration chromatography Weight molecular weight 185,000 149,000 128,000 average determined by SECMALLS (Reference) Very Very Good Pharmacological Effects in the Example good good 13 The same purified sodium alginate (lower endotoxin) used in Example 13 is Used for AL170, AL100 and AL4 0.

AL70: Kimica Corp., Mochida International Ltd., Sea Matrix (sterile), 1% viscosity: approximately 500 mPa.s.

AL100: Kimica Corp., sterilized, 1% viscosity: approximately 100 mPa.s.

AL40: FMC Biopolimer Inc., Pronova ™ SLM20 / 1% viscosity: approximately 30 mPa.s

(3) Discussion

As shown in Table 4, differences in molecular weights of three alginate types determined by SEC-MALLS were only observed in a range that did not definitely indicate a difference between them, and those values differed considerably from the measurement results obtained by filtration chromatography. in gel. As shown in Example 13, as there were well-defined differences in pharmacological effects between the samples used, 20 it was found that the molecular weights determined by gel filtration chromatography show a greater correlation with the therapeutic effects of alginates than the molecular weights determined. SEC-MALLS, and it has been found that molecular weights determined by gel filtration chromatography are suitable as parameters for specifying a preferable molecular weight range of alginates used in the cartilage regeneration composition or the treatment composition. of a cartilage disease.

Example 15

Effects of alginic acid on experimental arthritis pain

in rats

(1) Method

Mice with intra-articular arthritis induced by needle-shaped monosodium urate (MSU) crystals present with an abnormal gait caused by pain. An experimental model of arthritis pain in rats by MSU administration was prepared according to the method of Shizuhiko Ihara et al. (Folia Pharmacol. Japon, Vol. 100, pages 359-365 (1992)) to evaluate the effects of intraarticular administration of sodium alginate.

Males of Crl: CD mice were acquired at 5 weeks of age and used in the experiment after a one week acclimation period. 0.05 mL of a 5.0% saline suspension was injected into the right knee joint of the rats under anesthesia, followed by gait observation at 2, 4, 6 and 24 hours after injection. Gait was assessed by scores of one to five degrees consisting of normal gait (0 point), mild lameness (1 point), moderate lameness (2 points), walking on toes (3 points), and walking on three legs (4 points). Ten animals were distributed to each of the five groups indicated below:

A) Control group (saline dose group)

B) Dose group of 1% sodium hyaluronate solution

(ARTZ (Trademark), Kaken Pharmaceutical Co., Ltd., Molecular Weight: Approximately 900,000)

C) 2% Purified Sodium Alginate Solution Dose Group (Kimica Corp., Sterile, Molecular Weight:

approximately 1,000,000)

D) Purified sodium alginate 1% solution dose group (Sea Matrix (sterile), Kimica Corp., molecular weight: approximately 1,700,000)

E) Purified Sodium Alginate 2% Solution Dose Group (Sea Matrix (Sterile), Kimica Corp., Weight

molecular: approximately 1,700.0 00)

Fifty μΐ of each solution was administered at the same joint site one hour before MSU injection.

(2) Results and discussion

Time-based changes in gait scores are shown in FIG. 23. Gait scores for the 1% sodium hyaluronate solution dose group (group B) and the 2% sodium alginate solution dose groups 25 (CeE groups) were significantly lower than those in the control group. (group A), and pain suppression effects were observed. Dose-dependent pain suppression effects were observed in a comparison of 1% and 2% solutions containing sodium alginate with a molecular weight of about 1,700.00 (DeE groups). In addition, 2% sodium alginate solutions with molecular weights of 1,000,000 and 1,700,000 demonstrated equal pain suppression effects, although they had different viscosities of about 300 mPa.se and about 5,000 mPa.s. respectively.

In joints, MSU acts directly or indirectly

on synovial cells and neutrophils, and is believed to cause arthritis through cytokine production and the like (published by Shizuhiko Ihara, et al. above). Specifically, MSU induces pain as a result of the inflammatory reaction it induces. Sodium alginate solution demonstrated pain suppression effects in this model, and the observed effects were the same as those of sodium hyaluronate, which is used as a therapeutic drug for osteoarthritis and as joint pain suppression drug for chronic rheumatoid arthritis. A monovalent alginic acid metal salt has been confirmed to have effects that inhibit inflammation and pain, and is believed to be useful as a therapeutic drug for osteoarthritis, frozen shoulder, and the like, while being able to be applied to joint pain. associated with rheumatoid arthritis.

INDUSTRIAL APPLICABILITY As the cartilage regeneration composition of the present invention does not require excessive surgical procedure and can be injected into a cartilage trauma injury, the surgical procedure is simple and can be effectively promoted cartilage regeneration and particularly regeneration. hyaline cartilage, without placing an excessive burden on the body in terms of chondrocyte collection, periosteum, and the like. The cartilage regeneration composition of the present invention has gel curability upon contact with Ca Ions in the affected area. The composition may be retained in the affected area by curing the surface thereof by benefiting from this property. In the case of cells embedded for cartilage tissue regeneration in the cartilage regeneration composition of the present invention, the cells are readily dispersed in the cured gel. The composition can be used for various forms of cartilage lesions, and is capable of accommodating various application conditions.

The cartilage regeneration composition of the present invention is capable of demonstrating regenerative effects on hyaline cartilage, even without containing 15 cells because it contains a lower endotoxin monovalent metal salt of alginic acid. If it does not contain cells, the risk of virus and similar infection attributable to the body or the cultivation process may be reduced, thus making the procedure more

2 0 simple.

The composition for treating a cartilage disease of the present invention has cartilage repair effects, effects that suppress degenerative cartilage changes, cartilage protective effects, effects that inhibit joint tissue inflammation, and / or pain-suppressing effects. inflammation of joint tissue when injected into a joint in a liquid state, thereby allowing it to demonstrate therapeutic effects in a cartilage disease. The composition is

30 is particularly useful for the treatment of osteoarthritis, frozen shoulder treatment, and relief of joint pain associated with rheumatoid arthritis. 10

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Sequence Listing

<110> MOCHIDA PHARMACEUTICAL CO., LTD.

NATIONAL UNIVERSITY CORPORATION HOKKAIDO UNIVERSITY

<120> COMPOSITION FOR TREATMENT OF CARTILAGE DISEASES

<13 0> PCT08-0009 9

<150> JP 2007-41520

<151> 02/21/2007

<150> JP 2007-277005

<151> 10/24/2007

<160> 10 <170> PatentIn version 3 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <4 0 0> 1 taagagctcc aaggccaaga <210> 2 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <4 0 0> 2 tgtacctact cctttgaccg <21Q> 3 <211> 20 <212> DNA <213> Artificial Sequence <2 2 0> < 223> Synthetic DNA 10

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<400> 3 agagacctga actgggcaga <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 4 accacgatat gaggcacagt tt <210> 5 <211> 20 <212> DNA <213> Artificial string <220> <223> Synthetic DNA <400> 5 gccaggacct ccaggactat <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 6 ctttggacct gttgtccct <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <4 0 0> 7

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3 5 gtgcagttcg ccgggt 16

Claims (29)

Use of a lower monovalent alginic acid endotoxin metal salt, characterized in that it is used for the manufacture of a pharmaceutical composition which is used for the treatment of a cartilage disease, wherein the composition is adapted to be injected into a joint, and the composition does not contain cells for regeneration of cartilaginous tissue. Use according to claim 1, characterized in that said cartilage disease is osteoarthritis. Use according to claim 1, characterized in that said cartilage disease is frozen shoulder. Use according to claim 1, characterized in that said cartilage disease is joint pain associated with rheumatoid arthritis. Use according to claim 1, characterized in that said composition is used for the inhibition of degenerative cartilage changes. Use according to claim 1, characterized in that said composition is used for cartilage protection. Use according to claim 1, characterized in that said composition is used for cartilage repair. Use according to claim 1, characterized in that said composition is used for suppressing joint pain. Use according to claim 1, characterized in that said composition is used for inhibiting joint inflammation. Use according to claim 1, characterized in that said composition is used for the improvement of joint function. Use according to claim 1, characterized in that said composition is used for the regeneration of hyaline cartilage. Use according to claim 1, characterized in that a cross-linking agent is applied to the surface of the composition to cure the composition. Use according to claim 1, characterized in that the monovalent metal salt of alginic acid is sodium alginate. Use according to claim 13, characterized in that the sodium alginate is sodium alginate having a weight average molecular weight of 500,000 or more as determined by gel filtration chromatography. 15. Use of an alginic acid endotoxin lower monovalent metal salt, characterized in that it is used in the manufacture of a pharmaceutical composition which is used for the regeneration of hyaline cartilage, and which is applied to a cartilage trauma injury containing cells. - mesenchymal stem of the bone marrow, having a viscosity of 400 mPa.s to 5,000 mPa.s, and having fluidity. Use according to claim 15, characterized in that a cross-linking agent is applied to the surface of the composition to cure the composition. Use according to claim 15, characterized in that the cells are included in the composition after in vitro cultivation without inducing differentiation. Use according to claim 15, characterized in that the application to a cartilage trauma injury is: a) application to a cartilage defect, or b) formation of one or more holes in the cartilage trauma injury; cartilage or a cartilage defect and application to the formed holes. Use according to claim 15, characterized in that the composition is adhered for at least 5 seconds to a cartilage trauma injury having an opening or defect that is slanted or turned downward. Use according to claim 15, characterized in that the composition is applicable to cartilage trauma injury with a 16G needle. Use according to claim 15, characterized in that the monovalent metal salt of alginic acid is sodium alginate. 22. Composition for use in the treatment of a cartilage disease containing as an active ingredient therein a lower monovalent metal salt of alginic acid endotoxin, wherein the composition does not contain cells for regeneration of cartilaginous tissue. Composition according to Claim 22, characterized in that a cross-linking agent is applied to the surface of the composition to cure the composition. Composition according to Claim 22, characterized in that the monovalent metal salt of alginic acid is sodium alginate. Composition according to Claim 24, characterized in that the sodium alginate is sodium alginate having a weight average molecular weight of 500,000 or more as determined by gel filtration chromatography. 26. Composition for use in the regeneration of hyaline cartilage containing a monovalent lower metal salt of alginic acid endotoxin and bone marrow mesenchymal stem cells having a viscosity of 400 mPa.sa 5,000 mPa.s, and that have fluidity. Composition according to Claim 26, characterized in that a cross-linking agent is applied to the surface of the composition to cure the composition. Composition according to Claim 26, characterized in that the cells are included in the composition after in vitro cultivation without inducing differentiation. Composition according to Claim 26, characterized in that the monovalent metal salt of alginic acid is sodium alginate.